Peptide analogs of alpha-melanocyte stimulating hormone

ABSTRACT

Provided herein are stable peptide analogs of the native alpha-melanocyte stimulating hormone (α-MSH) having selectivity for the melanocortin 1 receptor (MC1R). Also provided herein are pharmaceutical preparations of the α-MSH peptide analogs, as well as methods of using these analogs in the treatment of medical and veterinary conditions involving MC1R.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/830,572, filed Aug. 19, 2015, now pending, which is a continuation ofU.S. application Ser. No. 13/890,039, filed May 8, 2013, now U.S. Pat.No. 9,115,174, which is a divisional of U.S. application Ser. No.12/408,560, filed Mar. 20, 2009, now U.S. Pat. No. 8,440,793, issued May14, 2013, which claims the priority benefit of U.S. provisionalapplication Ser. No. 61/056,373, filed May 27, 2008. The contents of theabove-listed applications are incorporated herein by this reference intheir entireties for all purposes.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 159792008102SeqList.txt,date recorded: Jul. 10, 2017, size: 28,719 bytes.

TECHNICAL FIELD

The invention relates to peptide analogs. In particular, the inventionrelates to peptide analogs of native alpha-melanocyte stimulatinghormone (α-MSH) having selectivity for the melanocortin 1 receptor(MC1R), pharmaceutical preparations thereof, as well as to the use ofthese analogs in the treatment of medical and veterinary conditions.

BACKGROUND ART

Neuropeptides are small biologically active peptides which are widelydistributed throughout the body and have functions from neurotransmitterto growth factor. Much evidence has indicated that neuropeptides haveanti-inflammatory capabilities. Among the many neuropeptides aremelanocortic peptides (melanocortins), which bind to and stimulatemelanocortin (MC) receptors. An example of a melanocortin includesα-melanocyte-stimulating hormone (α-MSH), mainly known for its abilityto regulate peripheral pigmentation, but is also known to haveanti-inflammatory and immunomodulatory capabilities. α-MSH neuropeptidehas been detected in several organs and is produced by neurons,pituitary, gut, skin, and immune cells.

The immunomodulatory capabilities of α-MSH have been demonstrated inmodels of contact hypersensitivity where hapten-specific tolerance wasinduced by injection with α-MSH and in suppressing bacterialendotoxin-mediated inflammation. Also, α-MSH has been shown to havetherapeutic activity in many animal disease models such as inflammatorybowel disease, arthritis, and experimental heart transplantation. Otheranimal models of brain inflammation, renal injury and liver inflammationhave demonstrated anti-inflammatory effects with this neuropeptide.α-MSH suppresses production of pro-inflammatory cytokines such as TNF-α,IL-6, and IL-1 and inhibits chemokines which reduce macrophage andneutrophil migration to inflammatory sites. Nitric oxide (NO) is acommon mediator for various forms of inflammation. NO synthesis byendotoxin-stimulated macrophages and neutrophils has also shown to beinhibited by α-MSH. In addition to its effects on cytokine production,α-MSH downregulates the expression of MHC class I, CD86 and CD40 onmonocytes and dendritic cells which effects antigen presentation andco-stimulation. α-MSH is also known to increase the formation ofinterleukin 10 (IL-10) in monocytes, which is believed to be animportant component in immunosuppressive effects.

Although the molecular mechanisms of the immunomodulatory effects ofα-MSH are not completely understood, a potential mechanism of action ofα-MSH is its ability to inhibit nuclear factor-κB activation in cells.The inhibition of NF-κB results in a suppression of pro-inflammatorycytokine production and nitric oxide synthesis by macrophages. α-MSHfunctions by binding specific receptors that belong to a group ofG-protein-coupled receptors with seven transmembrane domains. Thesereceptors include the melanocortin 1 and melanocortin 3 receptors (MCR-1and MCR-3) on macrophages by which the binding of α-MSH inhibits NF-κB.Many of the immunomodulatory effects of α-MSH are also mediated throughthe accumulation of cAMP. The binding of α-MSH to melanocortin receptorsincreases cAMP levels which may suppress the degradation of IκB andtherefore inhibit NF-κB translocation and nitric oxide production.

MC1 receptors, to which α-MSH bind and stimulates has been implicated invarious anti-inflammatory and immunomodulatory responses. Five types ofmelanocortin receptors have been identified, MC1-MC5. MC1-receptors havebeen found present on melanocytes, melanoma cells, macrophages,neutrophils, glioma cells, astrocytes, monocytes, endothelial cells, incertain areas of the brain, testis and ovary. There remains continuedinterest in compounds and methods to stimulate MC1-receptors and toproduce effective anti-inflammatory and immunomodulatory responses.

SUMMARY OF THE INVENTION

Provided herein is a substantially pure compound that selectively bindsmelanocortin 1 receptor (MC1R), said compound comprising a coretetrapeptide having the sequence His Xaa₁ Arg Trp (SEQ ID NO:1) or D-TrpD-Arg Xaa₂ D-His (SEQ ID NO:2); wherein Xaa₁ is D-Cha, D-Phe or Cha, andXaa₂ is D-Cha, D-Phe or Phe; or a pharmaceutically acceptable saltthereof. In some embodiments, the C-terminal sequence is D-Ser D-IleD-Ile D-Ser D-Ser (SEQ ID NO:3).

Further provided herein is a substantially pure compound thatselectively binds melanocortin 1 receptor (MC1R), said compoundcomprising a polypeptide (SEQ ID NO:9) having the sequence:

-   -   Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂        Xaa₁₃,    -   wherein Xaa₁ is D-Val, D-Ala or D-Lys;        -   Xaa₂ is D-Pro, D-Ala or D-Lys;        -   Xaa₃ is D-Lys, D-Orn, D-Nle, D-Ala or D-Lys;        -   Xaa₄ is Gly, or D-Ala;        -   Xaa₅ is D-Trp, Trp, D-3-benzothienyl-Ala, D-5-hydroxy-Trp,            D-5-methoxy-Trp, D-Phe, or D-Ala;        -   Xaa₆ is D-Arg, D-His, or D-Ala;        -   Xaa₇ is D-Cha, D-Phe, Phe, D-4-fluoro-Phg, D-3-pyridyl-Ala,            D-Thi, D-Trp, D-4-nitro-Phe, or D-Ala;        -   Xaa₈ is D-His, His, D-Arg, Phe, or D-Ala;        -   Xaa₉ is D-Glu, D-Asp, D-citrulline, D-Ser, or D-Ala;        -   Xaa₁₀ is D-Met, D-buthionine, D-Ile, or D-Ala;        -   Xaa₁₁ is D-Ser, D-Ile or D-Ala;        -   Xaa₁₂ is D-Tyr, D-Ser, or D-Ala;        -   Xaa₁₃ is D-Ser or D-Ala;    -   wherein no more than one Xaa₁₋₁₃ is D-Ala except when Xaa₁₋₃ are        all D-Ala, and no more than one Xaa₁₋₁₃ is an L-amino acid; or a        pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a substantially pure compoundcomprising a polypeptide having the sequence:

(SEQ ID NO: 4) D-Val D-Pro D-Lys Gly D-Trp D-Arg Phe D-His D-SerD-Ile D-Ile D-Ser D-Ser; (SEQ ID NO: 5)D-Val D-Pro D-Lys Gly D-Trp D-Arg D-Cha D-HisD-Ser D-Ile D-Ile D-Ser D-Ser; (SEQ ID NO: 6)Ser Tyr Ser Met Glu His Cha Arg Trp Gly Lys Pro Val; or (SEQ ID NO: 7)D-Val D-Pro D-Lys Gly D-Trp D-Arg D-Phe D-HisD-Glu D-Met D-Ser D-Tyr D-Ser;or a pharmaceutically acceptable salt thereof.

In some embodiments, the polypeptides provided herein are PEGylated.

In some embodiments, the compounds provided herein can be conjugated toa biologically active moiety.

In some embodiments, the compounds provided herein selectively bindMC1R. In some embodiments, the compounds exhibit at least one of thefollowing properties: ability to selectively activate MC1R, stability inplasma in vitro, and resistance to protease degradation.

In one aspect, provided herein is a pharmaceutical compositioncomprising any one of the substantially pure compounds provided hereinand a pharmaceutically acceptable excipient.

In another aspect, provided herein is a method of treating an autoimmunedisease or condition in a subject in need thereof, comprisingadministering to said subject a pharmaceutical composition comprising apharmaceutically acceptable excipient and a therapeutically effectiveamount of a compound provided herein. In some embodiments, theautoimmune disease or condition is selected from the group consisting ofmultiple sclerosis, diabetes type I, aplastic anemia, Grave's disease,coeliac disease, Crohn's disease, lupus, arthritis, osteoarthritis,autoimmune uveitis and myasthenia gravis.

In yet another aspect, provided herein is a method of treatinginflammation in a subject in need thereof comprising administering tosaid subject a pharmaceutical composition comprising a pharmaceuticallyacceptable excipient and a therapeutically effective amount of acompound provided herein. In some embodiments, the inflammation isassociated with a disease selected from the group consisting ofinflammatory bowel disease, rheumatoid arthritis, allergy,atherosclerosis, psoriasis, gastritis and ischemic heart disease.

In one aspect, provided herein is a method to reduce or inhibittransplant rejection in a subject in need thereof comprisingadministering to said subject a pharmaceutical composition comprising apharmaceutically acceptable excipient and a therapeutically effectiveamount of a compound provided herein.

In another aspect, provided herein is a method to treat melanoma in asubject in need thereof comprising administering to said subject apharmaceutical comprising a pharmaceutically acceptable excipient and atherapeutically effective amount of a compound provided herein.

In yet another aspect, provided herein is a method to treat melanoma ina subject in need thereof comprising administering to said subject apharmaceutical comprising a pharmaceutically acceptable excipient and atherapeutically effective amount of a conjugate comprising a compoundprovided herein that is conjugated to an anti-tumor payload. Theanti-tumor payload can be a radionuclide, a radiosensitizer, aphotosensitizer, a chemotherapeutic agent, or a toxin.

In a further aspect, provided herein is a kit comprising thepharmaceutical composition of a compound provided herein and optionallycomprising instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B show reduction of uveitis by native α-MSH. FIG. 1A shows datafrom B10.RIII mice were treated with native α-MSH (100 μg/mouse) IVdaily when clinical scores were 2-3. Uveitis was significantly reducedcompared with untreated control (p<0.01). FIG. 1B shows data fromB10.RIII mice were treated with native α-MSH (100 μg/mouse) IP ordexamethasone IP (0.2 mg/kg or 2.0 mg/kg) daily when clinical scoreswere 1-2 (n=5). Retinal inflammation was reduced after treatmentinitiation (p<0.05). Asterisk denotes significant differences fromcontrol.

FIGS. 2A-B illustrate amelioration of uveitis in late-stage disease byRI α-MSH and native α-MSH. EAU was induced in B10.RIII mice and 100μg/mouse native α-MSH, RI α-MSH or PBS was administered daily, i.v., onday 12 when mice reached late-stage disease (scores of 3). FIG. 2A showsdata from mice treated with native α-MSH or retro-RI α-MSH showedreduction in uveitis disease eye scores compared with PBS control mice.FIG. 2B shows individual maximum eye scores of mice in each group on day16 post-EAU induction (n=8). Asterisk denotes significant differencesbetween groups (p<0.05).

FIGS. 3A-D show retina images and individual eye scores from animalstreated with α-MSH or RI α-MSH. EAU was induced in B10.RIII mice, anddaily i.v. treatment with 100 μg/mouse of native α-MSH, RI α-MSH or PBSbegan on day 13 when mice reached late-stage disease. Fundoscopic imagesof retinas representing median eye scores from each group after 13 daysof treatment are shown (n=11). The PBS treated mouse with an eye scoreof 3 shows inflammatory lesions in several quadrants of the eye andvasculitis proximal to the optic nerve (FIG. 3A). The α-MSH and RI α-MSHtreated mice with eye scores of 1 show resolution of uveitis withinflammation only around the optic nerve (FIGS. 3B and 3C,respectively). Retinas represent median eye score from each group.Individual eye scores of mice in each group on day 13 post-treatment areshown in the graph (FIG. 3D). Asterisk denotes significant differencesbetween groups (p<0.01).

FIGS. 4A-C illustrate histopathology of RI α-MSH and native α-MSHtreated mice in EAU. Female B10.RIII mice were injected with IRBP+CFA toinduce EAU. Mice were treated daily, i.v., with 100 μg/mouse ofretro-inverso α-MSH, native α-MSH, or PBS when mice reached eye scoresof 3. The RI α-MSH or α-MSH treated group of mice ameliorated the ocularinflammatory response (p<0.05). Photographs show a hematoxylin and eosinstaining of eyes 10 days after the start of treatment representingmedian eye scores from PBS (FIG. 4A), α-MSH (FIG. 4B) and RI α-MSH (FIG.4C) treated groups of mice. Magnification is 100×.

FIG. 5 shows the effect of intraperitoneal daily dosing of retro-inversoα-MSH on uveitis compared with a scrambled peptide control. B10.RIIImice were treated daily by intraperitoneal injections with 100 μg of RIα-MSH or a scrambled D amino acid peptide control on day 11 afterdisease induction. Data show the clinical mean eye scores of RI α-MSH(n=4) and scrambled control peptide (n=5) treated groups of mice. Micewere treated a total of 13 days. Data is representative of twoexperiments. Asterisk denotes significant differences between groups(p<0.04).

FIG. 6 illustrates efficacy of RI α-MSH in the treatment of retinalinflammation. B10RIII mice were treated with RI α-MSH (3, 10, or 100μg/mouse) daily by IP injections when clinical scores were 4. Scrambledpeptide control was injected at 100 μg/mouse daily. Graph depictsclinical scores over time. Reduced retinal inflammation was observedafter treatment initiation with 100 or 10 μg/mouse. Limited beneficialclinical responses were observed in mice treated with a lower dose of RIα-MSH (3 μg/mouse) or with PBS or the control scrambled peptide (n=4).Asterisk denotes significant differences between groups (p<0.05).

FIGS. 7A-B show the effect of RI α-MSH on cAMP production in murinemelanoma cells. cAMP was measured in B16-F1 melanoma cells aftertreatment of cells with native α-MSH, retro-inverso α-MSH or scrambledcontrol peptide at concentrations 0.01 ng/ml-1000 ng/ml. FIG. 7A showsthat both native α-MSH and RI α-MSH significantly increased cAMP levels.Controls used to measure cAMP included forskolin at a 100 μMconcentration. FIG. 7B shows alanine scanning data. Alanine substitutedpeptides of RI α-MSH at 1 μg/ml were tested for increases in cAMP levelsin the murine B16-F1 melanoma cell line. Data is representative of twoexperiments. Sequence list may be found in Table 1. Asterisk denotessignificant differences between groups (p<0.01).

FIGS. 8A-B illustrate the disease course of MOG induced EAE. EAE wasinduced in C57BL/6 mice by injection of a MOG35-55 peptide (200μg/mouse) and CFA emulsion. Pertussis toxin was injected on day 0 andday 2. Daily i.p. treatment with 100 μg/mouse of α-MSH, RI α-MSH, or PBSstarted on Day 10. FIG. 8A shows clinical disease scores recorded daily.FIG. 8B shows individual disease scores in each group on day 20 afterdisease induction.

FIG. 9 shows the reduction of mean EAE disease scores by RI α-MSH. EAEwas induced in C57BL/6 mice by injections with an emulsion of MOG35-55peptide (200 μg/mouse) and CFA. Pertussis toxin was injected on day 0and day 2. Daily i.p. treatment with 100 μg or 30 μg/mouse of α-MSH orRI α-MSH, 2 mg/kg of dexamethasone, or PBS started on Day 10. Clinicaldisease scores were recorded daily.

FIGS. 10A-D illustrate the spinal cord histology of mice treated with RIα-MSH. EAE was induced in C57BL/6 mice by injection of a MOG35-55peptide (200 μg/mouse) and CFA emulsion. Pertussis toxin was injected onday 0 and day 2. Daily i.p. treatment with 100 μg/mouse of RI α-MSHbegan on Day 10. Spinal cord was collected on Day 24 after diseaseinduction. Two representative mice from each group are shown: PBStreated (FIGS. 10A and 10C) and RI α-MSH treated (FIGS. 10B and 10D).Arrows show sites of inflammatory cell infiltration.

FIGS. 11A-F show the amount of TNFα and IL-10 mRNA in the spleen of MOGpeptide primed mice during disease phase of EAE. Mice were primed with200 μg MOG peptide on day 0 and treated with PBS, α-MSH (100 μg) or RIα-MSH (100 μg) daily on days 10-15. Spleens were harvested on Days 1(FIGS. 11A and 11B), 4 (FIGS. 11C and 11D) and 7 (FIGS. 11E and 11F)after start of treatment and analyzed for TNFα and IL-10 mRNA expressionby quantitative PCR. Data show mean of 4 mice in each treatment group.RNA levels are normalized to β-actin.

FIGS. 12A-D illustrate the recall response to MOG 35-55 peptide. Micewere primed with 200 μg MOG35-55 peptide on day 0. On days 2-8 mice wereinjected i.p. with PBS, 100 μg α-MSH or 100 μg RI α-MSH (n=5). On day 9spleen (FIG. 12A) and lymph node (FIG. 12B) cells were harvested andstimulated with 25 μg/ml MOG35-55 peptide or OVA peptide in vitro. Cellswere pulsed with [3H] thymidine on day 3 in culture. Data show mean±SD.Supernatant was collected from spleen cell cultures stimulated with MOGpeptide after 24 hrs and cytokine levels of TNF-α and IFNγ (FIG. 12C)and MCP-1 (FIG. 12D) were analyzed by flow cytometry. Data show mean±SD.Naïve mice were not primed with MOG peptide.

FIGS. 13A-D illustrate cytokine profiles in serum and spleen after RIα-MSH treatment in MOG primed mice. Mice were primed with 200 μgMOG35-55 peptide on day 0. On days 2-8 mice were injected i.p. with PBS,100 μg α-MSH or 100 μg RI α-MSH (n=5). On day 9 spleen and serum wascollected. FIGS. 13A and 13B show TNF-α and IL-10 mRNA levels in thespleen, respectively, quantitated by real time PCR. Data show the meanof 4 mice in each treatment group. RNA levels are normalized to β-actin.Serum was also analyzed for cytokine levels by flow cytometry. FIG. 13Cshows TNF-α, MCP-1, IL-6 serum levels, and FIG. 13D shows IL-10 andIL-12 serum levels. Naïve mice were not primed with MOG peptide. Datashow mean±SD.

FIGS. 14A-B show the effect of α-MSH and RI α-MSH on macrophage markers.Mice were primed with MOG peptide in vivo and treated daily with α-MSHor RI α-MSH (100 μg/mouse) on days 1-7. Splenic macrophages wereanalyzed by flow cytometry for expression levels of CD14, CD40 and CD86.Cells were gated on either the CD11b⁺ (FIG. 14A) or F4/80⁺ (FIG. 14B)macrophage cell population. Data show both the mean percent positive ofthe gated macrophage population (n=5).

FIGS. 15A-B illustrate an LPS-induced increase of mMC1R mRNA levels inboth the peritoneal macrophages (FIG. 15A) and spleen (FIG. 15B).C57BL/6 mice (n=4) were injected i.p. with LPS (1 μg/mouse). Peritonealmacrophages and spleen were harvested at 0.5 hr, 1 hr, and 24 hr. mMC1RmRNA levels were quantitated by real time PCR. RNA levels werenormalized to 18 s.

FIGS. 16A-F show the effect of MSH is an in vivo LPS inflammation model.C57BL/6 mice were injected with 1 μg of LPS, i.p. After 30 min, micewere treated with Dexamethasone (2 mg/kg) and α-MSH (FIGS. 16A-16C) orRI α-MSH analog 891 (FIGS. 16D-16F), i.p. Serum was collected 2 hoursafter LPS challenge. The levels of TNF-α (FIGS. 16A and 16D), MCP-1(FIGS. 16B and 16E), and IL-10 (FIGS. 16C and 16F) were analyzed bycytometric bead assay by flow cytometry. Data show individual cytokinemeasurements and mean of each group (n=6).

FIGS. 17A-B illustrate stability of RI-α-MSH and α-MSH in plasma andserum. FIG. 17A shows RI-α-MSH and α-MSH peptide stability in plasma andPBS at 37° C. FIG. 17B shows serum half-life of RI-α-MSH and α-MSHpeptide after a single intravenous injection.

FIGS. 18A-B show the results of binding studies of MSH (FIG. 18A) andRI-MSH (FIG. 18B) to melanocortin receptors 1, 3, 4 and 5.

FIG. 19 shows inversion of the core tetrapeptide HfRW (variant disclosedin SEQ ID NO:1) and retroinverso MSH.

FIGS. 20A-C illustrates substitution of non-natural amino acid residuesat varying positions of RI-MSH.

FIG. 21 illustrates a representative competitive binding assay of RI-MSHand variations thereof in MC1R.

FIGS. 22A-D illustrate the effect of RI α-MSH analogs on cAMP levels inB16 F1 murine melanoma cells. FIG. 22A: Analogs 890, 891 and 892; FIG.22B: Analogs 893, 894 and 895; FIG. 22C: Analogs 880, 886 and 878; andFIG. 22D: Analogs 872, 878 and 869.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The α-MSH and MC1R proteins and nucleic acids of the present methods arenot limited to a particular source or species. Thus, the proteins andnucleic acids can be isolated or recombinant.

α-Melanocyte-stimulating hormone (α-MSH) is a polypeptide comprising thesequence Ser Tyr Ser Met Glu His Phe Arg Trp Gly Lys Pro Val (SEQ IDNO:8) (SYSMEHFRWGKPV). α-MSH has been recently examined foranti-inflammatory and immunomodulatory capabilities. α-MSH originatesfrom intracellular proteolytic cleavage of proopiomelanocortin hormone(POMC). α-MSH neuropeptide has been detected in several organs and isproduced by neurons, pituitary, gut, skin, and immune cells. α-MSH hasbeen reported to suppress effector T cell function, induce regulatory Tcells and have beneficial effects in autoimmune and transplant models.

“Conjugate” or “conjugate” includes two or more members which areattached, joined, coupled, complexed or otherwise associated with eachother. The members may be joined to each other through covalent bonds,ionic bonds, electrostatic, hydrogen boding, van der Waals interactionsor physical means.

“Biologically active” moieties include a molecule or compound thatelicits or modulates a physiological response. In one aspect, abiologically active compound stimulates melanocortin receptors,preferably MC1-receptors.

By “modulate” and “modulation” is meant that the activity of one or moreproteins or protein subunits is up regulated or down regulated, suchthat expression, level, or activity is greater than or less than thatobserved in the absence of the modulator. For example, the term“modulate” can mean “inhibit” or “stimulate”.

“C-terminal sequence” includes reference to the end of the amino acidchain terminated typically, but not necessarily, by a carboxyl group.The convention for writing peptide sequences is to put the C-terminalend on the right and write the sequence from N- to C-terminus. TheC-terminal sequence may comprise 1 to 100 amino acids, preferably 2 to15 amino acids, and even more preferably 3 to 10 amino acids. TheC-terminal sequence may terminate with a carboxyl group or the terminusmay be modified by well-known methods in the art to comprise afunctional member (e.g. targeting group, retention signal, lipid, andanchor).

The present invention provides a “substantially pure compound”. The term“substantially pure compound” is used herein to describe a molecule,such as a polypeptide (e.g., a polypeptide that binds MC1R, or afragment thereof) that is substantially free of other proteins, lipids,carbohydrates, nucleic acids, and other biological materials with whichit is naturally associated. For example, a substantially pure molecule,such as a polypeptide, can be at least 60%, by dry weight, the moleculeof interest. The purity of the polypeptides can be determined usingstandard methods including, e.g., polyacrylamide gel electrophoresis(e.g., SDS-PAGE), column chromatography (e.g., high performance liquidchromatography (HPLC)), and amino-terminal amino acid sequence analysis.

In one embodiment, the phrase “selectively binds” means that a compoundor polypeptide made or used in the present invention preferentiallybinds to one type of receptor over another type of receptor when in thepresence of a mixture of two or more receptors (e.g., melanocortinreceptors, MC1, MC2, MC3, MC4, MC5 receptors).

“Amino acid” or “amino acid sequence” include an oligopeptide, peptide,polypeptide, or protein sequence, or to a fragment, portion, or subunitof any of these, and to naturally occurring or synthetic molecules. Theterms “polypeptide” and “protein” include amino acids joined to eachother by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain modified amino acids other than the 20gene-encoded amino acids. The term “polypeptide” also includes peptidesand polypeptide fragments, motifs and the like. Capitalized,single-letter abbreviations of the amino acids refer to the naturalL-isomer. Lower case, single-letter abbreviations of the amino acidsdenotes the D-isomer.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably to refer to polymers of amino acids of any length.Peptides and polypeptides can be either entirely composed of synthetic,non-natural analogues of amino acids, or, is a chimeric molecule ofpartly natural peptide amino acids and partly non-natural analogs ofamino acids. In one aspect, a polypeptide is used in a composition, cellsystem or process of the invention (e.g., a host cell having a plasmidexpressing at least one enzyme of the invention). In addition,polypeptide can refer to compounds comprised of polymers of amino acidscovalently attached to another functional group (e.g., solubilizinggroup, a targeting group, PEG, non-amino acid group, or othertherapeutic agent).

Amino acids may be abbreviated using the following designation inparentheses: Proline (Pro), Valine (Val), Lysine (Lys), Ornithine (Orn),Norleucine (Nle), Glycine (Gly), Tryptophan (Trp), Alanine (Ala),Phenylalanine (Phe), Arginine (Arg), Histidine (His), Glutamic acid(Glu), Aspartic acid (Asp), Serine (Ser), Methionine (Met), Isoleucine(Ile), Tyrosine (Tyr), Cyclohexylalanine (Cha), 4-fluoro-D-phenylglycine(4-fluoro-D-Phg), 2-thienyl-D-alanine (D-Thi).

“Treatment”, “treating”, “treat” or “therapy” as used herein refers toadministering, to a mammal, agents that are capable of eliciting aprophylactic, curative or other beneficial effect in the individual.Treatment may additionally result in attenuating or ameliorating adisease or symptoms of a disease in a subject.

As used herein, the singular form “a”, “an”, and “the” includes pluralreferences unless indicated otherwise. For example, “a” target cellincludes one or more target cells.

Polypeptide compositions of the invention can contain any combination ofnon-natural structural components. Individual peptide residues can bejoined by peptide bonds, other chemical bonds or coupling means, suchas, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctionalmaleimides, N,N′-dicyclohexylcarbodiimide (DCC) orN,N′-diisopropylcarbodiimide (DIC). Linking groups that can be analternative to the traditional amide bond (“peptide bond”) linkagesinclude, e.g., ketomethylene (e.g., —C(═O)—CH₂— for —C(═O)—NH—),aminomethylene (CH₂—NH), ethylene, olefin (CH═CH), ether (CH₂—O),thioether (CH₂—S), tetrazole, thiazole, retroamide, thioamide, or ester(see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino Acids,Peptides and Proteins, Vol. 7, pp. 267-357, “Peptide BackboneModifications,” Marcel Dekker, NY, incorporated herein by reference).

Polypeptides used to practice the method of the invention can bemodified by either natural processes, such as post-translationalprocessing (e.g., phosphorylation, acylation, etc), or by chemicalmodification techniques, and the resulting modified polypeptides.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxylterminus. It will be appreciated that the same type of modification maybe present in the same or varying degrees at several sites in a givenpolypeptide. Also a given polypeptide may have many types ofmodifications. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphatidylinositol, cross-linkingcyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,PEGylation, proteolytic processing, phosphorylation, prenylation,selenoylation, sulfation, and transfer-RNA mediated addition of aminoacids to protein such as arginylation. See, e.g., Creighton, T. E.,Proteins—Structure and Molecular Properties 2nd Ed., W.H. Freeman andCompany, New York (1993); Posttranslational Covalent Modification ofProteins, B. C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983),incorporated herein by reference.

Additional Embodiments of Compounds

In some embodiments, the invention provided herein is a substantiallypure compound that selectively binds melanocortin 1 receptor (MC1R),said compound comprising a polypeptide (SEQ ID NO:10) having thesequence:

-   -   Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂        Xaa₁₃,    -   wherein Xaa₁ is D-Val, D-Ala or D-Lys;        -   Xaa₂ is D-Pro, D-Ala or D-Lys;        -   Xaa₃ is D-Lys, D-Orn, D-Nle, D-Ala or D-Lys;        -   Xaa₄ is Gly, or D-Ala;        -   Xaa₅ is D-Trp, Trp, D-3-benzothienyl-Ala, D-5-hydroxy-Trp,            D-5-methoxy-Trp, D-Phe, or D-Ala;        -   Xaa₆ is D-Arg, D-His, or D-Ala;        -   Xaa₇ is D-Cha, D-Phe, Phe, D-4-fluoro-Phg, D-3-pyridyl-Ala,            D-Thi, D-Trp, D-4-nitro-Phe, or D-Ala;        -   Xaa₈ is D-His, His, D-Arg, Phe, or D-Ala;        -   Xaa₉ is D-Glu, D-Asp, D-citrulline, D-Ser, or D-Ala;        -   Xaa₁₀ is D-Met, D-buthionine, D-Ile, or D-Ala;        -   Xaa₁₁ is D-Ser, D-Ile or D-Ala;        -   Xaa₁₂ is D-Tyr, D-Ser, or D-Ala;        -   Xaa₁₃ is D-Ser or D-Ala;            or a pharmaceutically acceptable salt thereof.

In some embodiments, the peptide analogs provided herein selectivelybinds MC1R and comprise a polypeptide (SEQ ID NO:11) having thesequence: Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂Xaa₁₃, wherein Xaa₁ is D-Val; Xaa₂ is D-Pro; Xaa₃ is D-Lys, D-Orn orD-Nle; Xaa₄ is Gly; Xaa₅ is D-Trp, Trp, D-3-benzothienyl-Ala,D-5-hydroxy-Trp, D-5-methoxy-Trp, or D-Phe; Xaa₆ is D-Arg or D-His; Xaa₇is D-Cha, D-Phe, Phe, D-4-fluoro-Phg, D-3-pyridyl-Ala, D-Thi, D-Trp, orD-4-nitro-Phe; Xaa₈ is D-His, His, D-Arg, Phe, or D-Ala; Xaa₉ is D-Glu,D-Asp, D-citrulline or D-Ser; Xaa₁₀ is D-Met, D-buthionine or D-Ile;Xaa₁₁ is D-Ser or D-Ile; Xaa₁₂ is D-Tyr or D-Ser; Xaa₁₃ is D-Ser;wherein no more than one Xaa₁₋₁₃ is an L-amino acid, or apharmaceutically acceptable salt thereof.

In other embodiments, the peptide analogs provided herein comprise apolypeptide (SEQ ID NO:12) having the sequence: Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃, wherein Xaa₁ is D-Val; Xaa₂is D-Pro; Xaa₃ is D-Lys, D-Orn or D-Nle; Xaa₄ is Gly; Xaa₅ is D-Trp orTrp; Xaa₆ is D-Arg; Xaa₇ is D-Cha, D-Phe, Phe or D-Thi; Xaa₈ is D-His orHis; Xaa₉ is D-Glu or D-Ser; Xaa₁₀ is D-Met, D-buthionine or D-Ile;Xaa₁₁ is D-Ser or D-Ile; Xaa₁₂ is D-Tyr or D-Ser; Xaa₁₃ is D-Ser;wherein no more than one Xaa₁₋₁₃ is an L-amino acid, or apharmaceutically acceptable salt thereof.

The compounds provided herein comprise peptide analogs of α-MelanocortinSimulating Hormone (α-MSH).

In one alternative embodiment, the peptide analogs consist of D-aminoacids. In other embodiments, the peptides comprise D amino acids, Lamino acids or a mixture of D and L amino acids. In other embodiments,the peptides are comprised of at least 40%, 50%, 60%, 70%, 80%, 90% or100% D amino acids. The compounds of the invention may also incorporatethe following non-limiting examples of non-standard amino acids:D-ornithine, D-norleucine, 3-benzothienyl-D-alanine, 5-hydroxy-D-Trp,5-methoxy-D-Trp, 4-fluoro-D-phenylglycine (4-fluoro-D-Phg),3-pyridyl-D-alanine, 2-thienyl-D-alanine (D-Thi), D-cyclohexylalanine(D-Cha), 4-nitro-D-Phe, D-citrulline, α-methyl-D-Met, and D-buthionine.

In some embodiments, the core tetrapeptide is comprised of the aminoacid sequence His Phe Arg Trp (variant described in SEQ ID NO:1) or TrpArg Phe His (variant described in SEQ ID NO:2), preferably in theD-amino acid configuration. In another embodiment, the core tetrapeptideis comprised of the amino acid sequence His D-Cha Arg Trp (variantdescribed in SEQ ID NO:1) or Trp Arg D-Cha His (variant described in SEQID NO:2), preferably in the D-amino acid configuration. In someembodiments, the core tetrapeptide is comprised of 4 D-amino acids. Inother embodiments, the core tetrapeptide has at least one non-standardamino acid.

The compounds provided herein can be synthetic or recombinant. Thecompounds of the invention may be manufactured by conventional chemicaltechniques known in the art. Methods of solid phase synthesis areexplained in published literature, such as, Solid Phase PeptideSynthesis: A Practical Approach (E. Atherton, et al. 1989). Thecompounds of the invention may also be manufactured by conventionalmolecular biological techniques known in the art. Within thisapplication, unless otherwise stated, definitions of the terms andillustration of the techniques of this application may be found in anyof several well-known references such as: Sambrook, J., et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress (1989); Goeddel, D., ed., Gene Expression Technology, Methods inEnzymology, 185, Academic Press, San Diego, Calif. (1991); “Guide toProtein Purification” in Deutshcer, M. P., ed., Methods in Enzymology,Academic Press, San Diego, Calif. (1989); Innis, et al., PCR Protocols:A Guide to Methods and Applications, Academic Press, San Diego, Calif.(1990); Freshney, R. I., Culture of Animal Cells: A Manual of BasicTechnique, 2nd Ed., Alan Liss, Inc. New York, N.Y. (1987); Murray, E.J., ed., Gene Transfer and Expression Protocols, pp. 109-128, The HumanaPress Inc., Clifton, N.J. and Lewin, B., Genes VI, Oxford UniversityPress, New York (1997). All of the cited references are fullyincorporated herein by reference.

In some embodiments, the peptide analogs provided herein selectivelybind or activate melanocortin 1 receptor (MC1R). Any suitable assay canbe employed to measure the binding or activation of MC1R. For example,cAMP induction in vitro can be used to assess MC1R activation. The invitro assessment can indicate activation in vivo. Additional embodimentsof this invention comprise any polypeptide selective for MC1-receptors.Identification of a selective MC1-receptor compound may be determined byan appropriate screening assay. A non-limiting example of a MC1-receptorbinding assay is disclosed in Example 4. In some embodiments, preferableMC1-receptors comprise a melanocortin 1 receptor from homo sapiens(GenBank Accession No: NP_002377).

Further embodiments of the invention are directed to compounds thatmodulate cAMP, nitric oxide (NO), TNF-α, TNF-α mRNA, IL-10 mRNA, IL-10,IFNγ, IL-6, IL-12 and/or MCP-1 levels. In some embodiments, thecompounds increase cAMP levels. In other embodiments, the compounds aredirected to the decrease or inhibition of levels of nitric oxide (NO),TNF-α, TNF-α mRNA, IL-10 mRNA, IL-10, IFNγ, IL-6, IL-12 and/or MCP-1.The identity of compounds that modulate abovementioned levels may bedetermined through screening assays. Acceptable assays known in the artcan be used to measure the abovementioned levels. Non-limiting examplesof assays for identifying compounds that exhibit desirable modulation ofthese levels are disclosed in the examples.

In some embodiments, the compounds of this invention may modulate immuneresponse and inflammation by an alternative mechanism of action and isnot limited to the mechanisms disclosed herein.

In some embodiments, the peptides provided herein have improved plasmastability and resistance to protease degradation. Plasma stability andresistance to protease degradation can be assessed by any suitablemethod. A non-limiting example has been disclosed in Example 19. The invitro assessment can indicate performance, improved resistance and alonger half-life in vivo.

The methods provided herein can be practiced in vivo, ex vivo or invitro.

PEGylated Peptides

In some embodiments, the peptides are modified to enhance the half-lifeof the peptide. In some embodiments, the peptide is PEGylated. In someembodiments, PEGylated peptides are directed to peptides covalentlyattached or conjugated to one or more polyethylene glycol (PEG) polymerchains. PEG polymer chains may include modified, functionalized orotherwise derivatized PEG chains. In another embodiment, the PEG polymerchain may have at least one or more branch points. In some preferredembodiments, PEG polymer chains and the corresponding PEGylated peptideare water soluble, are highly mobile in solution, lack toxicity andimmunogenicity, have ready clearance from the body and may have altereddistribution in the body. In some preferred embodiments, thepharmacokinetic nature of the PEGylated peptide is modulated by the typeof PEG chain. Strategies and methods for the preparation of PEGylatedpeptides are carried out by methods known in the art (G. Pasuta and F.M. Veronese (2007) “Polymer-drug conjugation, recent achievements andgeneral strategies” Progress in Polymer Science 32(8-9): 933-961,incorporated herein by reference). First and second generationPEGylation of protein processes may be found in the art.

A non-limiting example of PEGylation comprises the first step of thesuitable functionalization of the PEG polymer at one or both terminals(for linear PEGs). PEGs that are activated at each terminus with thesame reactive moiety is known as “homobifunctional”, where as if thefunctional groups present are different, then the PEG derivative isreferred as “heterobifunctional” or “heterofunctional.” The chemicallyactive or activated derivatives of the PEG polymer are prepared toattach the PEG to the desired molecule. The choice of the suitablefunctional group for the PEG derivative is based on the type ofavailable reactive group on the molecule that will be coupled to thePEG. Non-limiting examples of reactive amino acids include lysine,cysteine, histidine, arginine, aspartic acid, glutamic acid, serine,threonine and tyrosine. The N-terminal amino group and the C-terminalcarboxylic acid can also be used.

Other heterobifunctional PEGs for conjugation: These heterobifunctionalPEGs are very much useful in linking two entities, where a hydrophilic,flexible and biocompatible spacer is needed. Preferred end groups forheterobifunctional PEGs are maleimide, vinyl sulphones, pyridyldisulphide, amine, carboxylic acids and N-hydroxysuccinimide (NHS)esters.

In some embodiments of the invention, the PEGylated peptide may have amolecular weight range between 0.2 kDa-100 kDa. In some preferredembodiments of the invention, the PEGylated peptide may have a molecularweight range between 0.2 kDa-40 kDa. In some preferred embodiments ofthe invention, the PEGylated peptide may have a molecular weight rangebetween 0.2 kDa-15 kDa. In other embodiments, the preferred averagemolecular weight (in Da; as determined by size exclusion chromatography)for commercially available PEGs, can be selected from <1 k, 2 k, 3.5 k,5 k, 10 k, 20 k, 30 k, 40 k, and above but can be any MW depending uponthe pharmacokinetics desired. For example, lower molecular weightheterobifunctional PEGs which may be used as linkers and lower molecularweight PEGs may be used to improve peptide solubility. The PEG also canbe multi-arm, forked, or branched.

In some embodiments, the peptide is conjugated to targeting molecules orfunction as a targeting molecule, wherein the targeting moleculepreferentially associates with a desired receptor. In some preferredembodiments of the invention the peptide is conjugated to a cytotoxicagent. The term “cytotoxic agent” refers to a substance that inhibits orprevents the expression activity of cells, function of cells and/orcauses destruction of cells. The term is intended to include radioactiveisotopes chemotherapeutic agents, and toxins such as small moleculetoxins or enzymatically active toxins of bacterial, fungal, plant oranimal origin, including fragments and/or variants thereof. Non-limitingexamples of the cytotoxic agent include maytansine, dolastatin and itsanalogs including tasidotin and auristatin. In other embodiments of theinvention, non-limiting examples of cytotoxic agents include, but arenot limited to maytansinoids, yttrium, bismuth, ricin, ricin A-chain,saporin, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin, diphtheria toxin subunit A, truncatedPseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin Achain, alpha-sarcin, gelonin, mitogellin, restrictocin, phenomycin,enomycin, curicin, crotin, calicheamicin, sapaonaria officinalisinhibitor, and glucocorticoid and other chemotherapeutic agents, as wellas radioisotopes such as At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³² and radioactive isotopes of Lu. Antibodies may also beconjugated to an anti-cancer pro-drug activating enzyme capable ofconverting the pro-drug to its active form.

The peptide may be linked either directly or indirectly to the cytotoxicor targeting agent by any method presently known in the art. In somepreferred embodiments of the invention, the peptide is tethered to thecytotoxic agent or targeting agent by one or more linkers to form aconjugate, so long as the inclusion of the linker does not substantiallyimpede the function, binding, toxicity or inclusion of the peptide orconjugated agent.

Non-limiting examples of linkers include ionic and covalent bonds andany other sufficiently stable association, whereby the targeted agentwill be internalized by a cell to which the conjugated is targeted. Thelinker moiety is selected depending upon the properties desired.Considerations of linker selection may include the relief or decrease ofsteric hindrance caused by proximity of the conjugated elements, thealteration of other properties of the conjugate, such as thespecificity, toxicity, solubility, serum stability and/or intracellularavailability of the conjugate and/or to increase the flexibility of thelinkage. The linker may be any type of linkage and examples aredescribed in U.S. Pat. Nos. 7,166,702 and 5,194,425, both of which arefully incorporated herein by reference.

Linkers and linkages that are suitable for chemically linked conjugatesinclude, but are not limited to, disulfide bonds, thioether bonds,hindered disulfide bonds, and covalent bonds between free reactivegroups, such as amine and thiol groups. These bonds are produced usingheterobifunctional reagents to produce reactive thiol groups on one orboth of the polypeptides and then reacting the thiol groups on onepolypeptide with reactive thiol groups or amine groups to which reactivemaleimido groups or thiol groups can be attached on the other. Otherlinkers include, acid cleavable linkers, such as bismaleimidoethoxypropane, acid labile-transferrin conjugates and adipic acid dihydrazide,that would be cleaved in more acidic intracellular compartments; crosslinkers that are cleaved upon exposure to UV or visible light andlinkers, such as the various domains, such as CH1, CH2, and CH3, fromthe constant region of human IgG1 (see Batra et al. (1993) Mol. Immunol.30:379-386, incorporated herein by reference).

Chemical linkers and peptide linkers may be inserted by covalentlycoupling the linker to the peptide and the targeting or cytotoxic agent.The heterobifunctional agents, described below, may be used to effectsuch covalent coupling.

Heterobifunctional Cross-Linking Reagents

Numerous heterobifunctional cross-linking reagents that are used to formcovalent bonds between amino groups and thiol groups and to introducethiol groups into proteins, are known to those of skill in this art(see, e.g., the PIERCE CATALOG, Immuno Technology Catalog & Handbook,1992-1993, which describes the preparation of and use of such reagentsand provides a commercial source for such reagents; see also Cumber etal. (1992) Bioconjugate Chem. 3′:397-401; Thorpe et al. (1987) CancerRes. 47:5924-5931; Gordon et al. (1987) Proc. Natl. Acad. Sci. USA84:308-312; Walden et al. (1986) J. Mol. Cell Immunol. 2:191-197;Carlsson et al. (1978) Biochem. J. 173: 723-737; Mahan et al. (1987)Anal. Biochem. 162:163-170; Wawryznaczak et al. (1992) Br. J. Cancer66:361-366; Fattom et al. (1992) Infection & Immun. 60:584-589). All ofthe cited references are fully incorporated herein by reference. Thesereagents may be used to form covalent bonds between the targeting agent,the chemokine, and the targeted agent. These reagents include, but arenot limited to: N-succinimidyl-3-(2-pyridyidithio)propionate (SPDP;disulfide linker); sulfosuccinimidyl6-[3-(2-pyridyldithio)propionamido]hexanoate (sulfo-LC-SPDP);succinimidyloxycarbonyl-α-methyl benzyl thiosulfate (SMBT, hindereddisulfate linker); succinimidyl6-[3-(2-pyridyidithio)propionamido]hexanoate (LC-SPDP);sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC); succinimidyl 3-(2-pyridyldithio)butyrate (SPDB; hindereddisulfide bond linker); sulfosuccinimidyl2-(7-azido-4-methylcoumarin-3-acetamide) ethyl-1,3-dithiopropionate(SAED); sulfo-succinimidyl 7-azido-4-methylcoumarin-3-acetate (SAMCA);sulfosuccinimidyl6-[alpha-methyl-alpha-(2-pyridyidithio)toluamido]hexanoate(sulfo-LC-SMPT); 1,4-di-[3′-(2′-pyridyidithio)propionamido]butane(DPDPB); 4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridylthio)toluene(SMPT, hindered disulfate linker); sulfosuccinimidyl6[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-SMPT);m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS);m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS);N-succinimidyl(4-iodoacetyl)aminobenzoate (SLAB; thioether linker);sulfosuccinimidyl(4-iodoacetyl)amino benzoate (sulfo-SIAB);succinimidyl-4-(p-maleimidophenyl)butyrate (SMPB);sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-SMPB);azidobenzoyl hydrazide (ABH).

Other heterobifunctional cleavable cross-linkers include, N-succinimidyl(4-iodoacetyl)-aminobenzoate; sulfosuccinimidyl(4-iodoacetyl)-aminobenzoate;4-succinimidyl-oxycarbonyl-a-(2-pyridyldithio)-toluene;sulfosuccinimidyl-6-[α-methyl-α-(pyridyldithiol)-toluamido]hexanoate;N-succinimidyl-3-(−2-pyridyidithio)-proprionate; succinimidyl6[3(−(−2-pyridyldithio)-proprionamido]hexanoate; sulfosuccinimidyl6[3(−(−2-pyridyldithio)-propionamido]hexanoate;3-(2-pyridyidithio)-propionyl hydrazide, Ellman's reagent,dichlorotriazinic acid, S-(2-thiopyridyl)-L-cysteine. Further exemplarybifunctional linking compounds are disclosed in U.S. Pat. Nos.5,349,066, 5,618,528, 4,569,789, 4,952,394 and 5,137,877, all of whichare fully incorporated herein by reference.

Acid Cleavable, Photocleavable and Heat Sensitive Linkers

Acid cleavable linkers, photocleavable and heat sensitive linkers mayalso be used, particularly where it may be necessary to cleave thetargeted agent to permit it to be more readily accessible to reaction.Acid cleavable linkers include, but are not limited to,bismaleimidoethoxy propane; and adipic acid dihydrazide linkers (see,e.g., Fattom et al. (1992) Infection & Immun. 60:584-589, incorporatedherein by reference) and acid labile transferrin conjugates that containa sufficient portion of transferrin to permit entry into theintracellular transferrin cycling pathway (see, e.g., Welhöner et al.(1991) J. Biol. Chem. 266:4309-4314, incorporated herein by reference).

Photocleavable linkers are linkers that are cleaved upon exposure tolight (see, e.g., Goldmacher et al. (1992) Bioconj. Chem. 3:104-107,which linkers are herein incorporated by reference), thereby releasingthe targeted agent upon exposure to light. Photocleavable linkers thatare cleaved upon exposure to light are known (see, e.g., Hazum et al.(1981) in Pept., Proc. Eur. Pept. Symp., 16th, Brunfeldt, K (Ed), pp.105-110, which describes the use of a nitrobenzyl group as aphotocleavable protective group for cysteine; Yen et al. (1989)Makromol. Chem. 190:69-82, which describes water soluble photocleavablecopolymers, including hydroxypropylmethacrylamide copolymer, glycinecopolymer, fluorescein copolymer and methylrhodamine copolymer;Goldmacher et al. (1992) Bioconj. Chem. 3:104-107, which describes across-linker and reagent that undergoes photolytic degradation uponexposure to near UV light (350 nm); and Senter et al. (1985) Photochem.Photobiol. 42:231-237, which describes nitrobenzyloxycarbonyl chloridecross linking reagents that produce photocleavable linkages), therebyreleasing the targeted agent upon exposure to light. All of the citedreferences are fully incorporated herein by reference. Such linkerswould have particular use in treating dermatological or ophthalmicconditions that can be exposed to light using fiber optics. Afteradministration of the conjugate, the eye or skin or other body part canbe exposed to light, resulting in release of the targeted moiety fromthe conjugate. Such photocleavable linkers are useful in connection withdiagnostic protocols in which it is desirable to remove the targetingagent to permit rapid clearance from the body of the animal.

Other Linkers for Chemical Conjugation

Other linkers, include trityl linkers, particularly, derivatized tritylgroups to generate a genus of conjugates that provide for release oftherapeutic agents at various degrees of acidity or alkalinity. Theflexibility thus afforded by the ability to pre-select the pH range atwhich the therapeutic agent will be released allows selection of alinker based on the known physiological differences between tissues inneed of delivery of a therapeutic agent (see, e.g., U.S. Pat. No.5,612,474, incorporated herein by reference). For example, the acidityof tumor tissues appears to be lower than that of normal tissues.

Peptide Linkers

The linker moieties can be peptides. Peptide linkers can be employed inthe conjugate. The peptide linker typically a has from about 2 to about60 amino acid residues, for example from about 5 to about 40, or fromabout 10 to about 30 amino acid residues. The length selected willdepend upon factors, such as the use for which the linker is included.

The linker moiety can be a flexible spacer amino acid sequence, such asthose known in single-chain antibody research. Examples of such knownlinker moieties include, but are not limited to, GGGGS (SEQ ID NO:13),(GGGGS)n (SEQ ID NO:13), GKSSGSGSESKS (SEQ ID NO:14), GSTSGSGKSSEGKG(SEQ ID NO:15), GSTSGSGKSSEGSGSTKG (SEQ ID NO:16), GSTSGSGKSSEGKG (SEQID NO:17), GSTSGSGKPGSGEGSTKG (SEQ ID NO:18), EGKSSGSGSESKEF (SEQ IDNO:19), SRSSG (SEQ ID NO:20), SGSSC (SEQ ID NO:21). A Diphtheria toxintrypsin sensitive linker having the sequenceAMGRSGGGCAGNRVGSSLSCGGLNLQAM (SEQ ID NO:22) is also useful.

Additional linking moieties are described, for example, in Huston etal., Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883, 1988; Whitlow, M., etal., Protein Engin. 6:989-995, 1993; Newton et al., Biochemistry35:545-553, 1996; A. J. Cumber et al., Bioconj. Chem. 3:397-401, 1992;Ladurner et al., J. Mol. Biol. 273:330-337, 1997; and U.S. Pat. No.4,894,443, all of which publications are incorporated herein byreference.

Other linkers include, but are not limited to: enzyme substrates, suchas cathepsin B substrate, cathepsin D substrate, trypsin substrate,thrombin substrate, subtilisin substrate, Factor Xa substrate, andenterokinase substrate; linkers that increase solubility, flexibility,and/or intracellular cleavability include linkers, such as (glymser)nand (sermgly)n, (see, e.g., PCT Pub. No. WO 96/06641, incorporatedherein by reference, which provides exemplary linkers for use inconjugates). In some embodiments, several linkers may be included inorder to take advantage of desired properties of each linker.

Preparation of Conjugates

Conjugates with linked targeted agents can be prepared either bychemical conjugation, recombinant DNA technology, or combinations ofrecombinant expression and chemical conjugation. The peptide of theinvention and the cytotoxic or targeting agent may be linked in anyorientation and more than one targeting agent and/or targeted agent maybe present in a conjugate.

In some preferred embodiments of the invention, the cytotoxic agent istethered to the peptide by means of a hydrophilic and biocompatiblespacer polymer, including a short alkyl chain, polysialic or hyaluronicacid, a polypeptide or a PEG. In some preferred embodiments of theinvention, the cytotoxic agent is conjugated through a cleavable linker,e.g., a disulfide linkage or peptide containing a sequence cleavable bylysosomal proteases such as cathepsins. In some preferred embodiments ofthe invention, the spacer is attached either to the N- or C-terminus ofthe peptide.

Formulations

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Pharmaceutical formulations can be prepared according to any methodknown to the art for the manufacture of pharmaceuticals. Suchformulations can contain sweetening agents, flavoring agents, coloringagents and preserving agents. A formulation can be admixed with nontoxicpharmaceutically acceptable excipients which are suitable formanufacture. Such an “excipient” generally refers to a substantiallyinert material that is nontoxic and does not interact with othercomponents of the composition in a deleterious manner. Pharmaceuticallyacceptable excipients include, but are not limited to, liquids such aswater, buffered saline, polyethylene glycol, hyaluronic, glycerol andethanol. Pharmaceutically acceptable salts can be included therein, forexample, mineral acid salts such as trifluoroacetate, hydrochlorides,hydrobromides, phosphates, sulphates, and the like; and the salts oforganic acids such as acetates, propionates, malonates, benzoates, andthe like.

The therapeutic agent may be administered in a medicament orpharmaceutical composition suitable for delivery. Formulations maycomprise one or more diluents, emulsifiers, preservatives, buffers,excipients, etc., and may be provided in such forms as lyophilizedpowders, sprays, creams, lotions, gels, on patches, in implants, etc.Pharmaceutical formulations for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art inappropriate and suitable dosages. Such carriers enable thepharmaceuticals to be formulated in unit dosage forms as tablets, pills,powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries,suspensions, etc., suitable for ingestion by the patient. Pharmaceuticalpreparations for oral use can be formulated as a solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable additional compounds, if desired, toobtain tablets or dragée cores. Suitable solid excipients arecarbohydrate or protein fillers include, e.g., sugars, includinglactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,potato, or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; andgums including arabic and tragacanth; and proteins, e.g., gelatin andcollagen. Disintegrating or solubilizing agents may be added, such asthe cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Aqueous suspensions can contain an active agent (e.g., a chimericpolypeptide or peptidomimetic of the invention) in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethylene oxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol (e.g.,polyoxyethylene sorbitol mono-oleate), or a condensation product ofethylene oxide with a partial ester derived from fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). Theaqueous suspension can also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

Oil-based pharmaceuticals are useful for administration of hydrophobicactive agents of the invention. Oil-based suspensions can be formulatedby suspending an active agent (e.g., a chimeric composition of theinvention) in a vegetable oil, such as arachis oil, olive oil, sesameoil or coconut oil, or in a mineral oil such as liquid paraffin; or amixture of these. See e.g., U.S. Pat. No. 5,716,928, incorporated hereinby reference, describing using essential oils or essential oilcomponents for increasing bioavailability and reducing inter- andintra-individual variability of orally administered hydrophobicpharmaceutical compounds (see also U.S. Pat. No. 5,858,401, incorporatedherein by reference). The oil suspensions can contain a thickeningagent, such as beeswax, hard paraffin or cetyl alcohol. Sweeteningagents can be added to provide a palatable oral preparation, such asglycerol, sorbitol or sucrose. These formulations can be preserved bythe addition of an antioxidant such as ascorbic acid. As an example ofan injectable oil vehicle, see Minto (1997) J. Pharmacol. Exp. Ther.281:93-102, incorporated herein by reference. The pharmaceuticalformulations of the invention can also be in the form of oil-in-wateremulsions. The oily phase can be a vegetable oil or a mineral oil,described above, or a mixture of these. Suitable emulsifying agentsinclude naturally-occurring gums, such as gum acacia and gum tragacanth,naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan mono-oleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. Theemulsion can also contain sweetening agents and flavoring agents, as inthe formulation of syrups and elixirs. Such formulations can alsocontain a demulcent, a preservative, or a coloring agent.

It is also contemplated that a composition or medicament comprising thetherapeutic agent can contain a pharmaceutically acceptable carrier thatserves as a stabilizer, particularly for peptide, protein,polynucleotide or other like agents. Examples of suitable carriers thatalso act as stabilizers for peptides include, without limitation,pharmaceutical grades of dextrose, sucrose, lactose, trehalose,mannitol, sorbitol, inositol, dextran, and the like. Other suitablecarriers include, again without limitation, starch, cellulose, sodium orcalcium phosphates, citric acid, tartaric acid, glycine, high molecularweight polyethylene glycols (PEGs), and combination thereof. It may alsobe useful to employ a charged lipid and/or detergent. Suitable chargedlipids include, without limitation, phosphatidylcholines (lecithin), andthe like. Detergents will typically be a nonionic, anionic, cationic oramphoteric surfactant. Examples of suitable surfactants include, forexample, TERGITOL® and TRITON® surfactants (Union Carbide Chemicals andPlastics, Danbury, Conn.), polyoxyethylenesorbitans, for example, TWEEN®surfactants (Atlas Chemical Industries, Wilmington, Del.),polyoxyethylene ethers, for example Brij, pharmaceutically acceptablefatty acid esters, for example, lauryl sulfate and salts thereof (SDS),and like materials. A thorough discussion of pharmaceutically acceptableexcipients, carriers, stabilizers and other auxiliary substances isavailable in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J.1991), incorporated herein by reference.

Pharmaceutical compositions of the present invention suitable forparenteral administration comprise one or more compounds of theinvention in combination with one or more pharmaceutically-acceptablesterile isotonic aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, or sterile powders which may be reconstitutedinto sterile injectable solutions or dispersions just prior to use,which may contain sugars, alcohols, antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient, or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the compounds of the present inventionmay be ensured by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, such assugars, sodium chloride, phosphate buffered saline, and the like intothe compositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

In the methods of the invention, the pharmaceutical compounds can alsobe delivered as microspheres for slow release in the body. For example,microspheres can be administered via intradermal injection of drug whichslowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polym.Ed. 7:623-645; as biodegradable and injectable gel formulations, see,e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres fororal administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol.49:669-674. All of the cited references are fully incorporated herein byreference

Injectable depot forms are made by forming microencapsule matrices ofthe compounds of the present invention in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

The polypeptide may be administered on its own or in combination withanother therapeutic compound. In particular, the polypeptide may beadministered in conjunction with a therapeutic compound used to treatmelanoma, inflammation or an autoimmune disease in the mammal. Thepolypeptide and additional therapeutic compound may be formulated in thesame or different compositions. The polypeptide may be administeredsimultaneously, sequentially or separately from the additionaltherapeutic compound.

Dosage

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. When thecompounds of the present invention are administered as pharmaceuticals,to humans and animals, they can be given per se or as a pharmaceuticalcomposition containing, for example, 0.1 to 99% of active ingredient,more preferably, 10 to 30%, in combination with a pharmaceuticallyacceptable carrier.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion or metabolism of the particularcompound being employed, the rate and extent of absorption, the durationof the treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, oral, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thepresent invention for a patient, when used for the indicated effects,will have a non-limiting range from about 1 mcg to about 5 mg perkilogram of body weight per hour. In other embodiments, the dose willhave a non-limiting range from about 5 mcg to about 2.5 mg per kilogramof body weight per hour. In further embodiments, the dose will have anon-limiting range from about 5 mcg to about 1 mg per kilogram of bodyweight per hour.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In one embodiment, the compound isadministered as one dose per day. In further embodiments, the compoundis administered continuously, as through intravenous or other routes. Inother embodiments, the compound is administered less frequently thandaily, such as weekly or less.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition).

The subject receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as rabbits,equines, cattle such as bovine, swine, goat and sheep; and poultry andpets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable carriers and can also beadministered in conjunction with antimicrobial agents such aspenicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

Possible Routes of Administration for Disclosed Compounds

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration. As used herein, theterm “route” of administration is intended to include, but is notlimited to subcutaneous injection, intravenous injection, intraocularinjection, intradermal injection, intramuscular injection,intraperitoneal injection, intratracheal administration, intraadiposaladministration, intraarticular administration, intrathecaladministration, epidural administration, inhalation, intranasaladministration, oral administration, sublingual administration, buccaladministration, rectal administration, vaginal administration,intracisternal administration, transdermal administration and topicaladministration, or administration via local delivery (for example bycatheter or stent). The compounds may also be administered orco-administered in slow release dosage forms.

In the methods of the invention, the pharmaceutical compounds can alsobe administered by in intranasal, intraocular, periocular andintravaginal routes including suppositories, insufflation, powders andaerosol formulations (for examples of steroid inhalants, see Rohatagi(1995) J. Clin. Pharmacol. 35:1187-1193; Tjwa (1995) Ann. Allergy AsthmaImmunol. 75:107-111, incorporated herein by reference). Suppositoriesformulations can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at body temperatures and will therefore melt in the body torelease the drug. Such materials are cocoa butter and polyethyleneglycols.

In the methods of the invention, the pharmaceutical compounds can bedelivered transdermally, by a topical route, formulated as applicatorsticks, solutions, suspensions, emulsions, gels, creams, ointments,pastes, jellies, powders, and aerosols.

In the methods of the invention, the pharmaceutical compounds can beparenterally administered, such as by intravenous (IV) administration oradministration into a body cavity (e.g., the synovial space) or lumen ofan organ. These formulations can comprise a solution of active agentdissolved in a pharmaceutically acceptable carrier. Acceptable vehiclesand solvents that can be employed are water and Ringer's solution, anisotonic sodium chloride. In addition, sterile fixed oils can beemployed as a solvent or suspending medium. For this purpose any blandfixed oil can be employed including synthetic mono- or diglycerides.

As described herein, the methods of the present invention may be usedalone or in combination with other approaches for the treatment of anautoimmune disease or the other conditions described herein.

The particular combination of therapies (therapeutics or procedures) toemploy in a combination regimen will take into account compatibility ofthe desired therapeutics and/or procedures and the desired therapeuticeffect to be achieved. It will also be appreciated that the therapiesemployed may achieve a desired effect for the same disorder (forexample, an inventive compound may be administered concurrently withanother agent used to treat the same disorder), or they may achievedifferent effects (e.g., control of any adverse effects). As usedherein, additional therapeutic agents that are normally administered totreat or prevent a particular disease or condition, are known as“appropriate for the disease, or condition, being treated”.

A combination treatment of the present invention as defined herein maybe achieved by way of the simultaneous, sequential or separateadministration of the individual components of said treatment.

Therapeutic Applications

Administration of the polynucleotide or a modulatory compound(hereinafter “therapeutic agent”) as discussed herein may be either forpreventative or therapeutic purpose. When provided preventatively, thetherapeutic agent is provided in advance of any symptoms. Thepreventative administration of the therapeutic agent serves to preventor attenuate any symptoms. When provided therapeutically, thetherapeutic agent is provided at (or shortly after) the onset of asymptom of the disease or disorder. The therapeutic administration ofthe therapeutic agent serves to attenuate any actual exacerbation of thesymptoms.

The individual treated may be any mammal. In one aspect, the mammal is ahuman. In another aspect, the human has an autoimmune disease orcondition. In other aspects, the autoimmune disease or condition isassociated with or selected from the group consisting of multiplesclerosis, diabetes type I, aplastic anemia, Grave's disease, coeliacdisease, Crohn's disease, lupus, arthritis, osteoarthritis, autoimmuneuveitis, autoimmune encephalomyelitis, and myasthenia gravis.

In other aspects, the human has an inflammation disease or condition. Inanother aspect, the inflammation disease or condition is associated withor selected from the group consisting of inflammatory bowel disease,rheumatoid arthritis, allergy, atherosclerosis, psoriasis, gastritis andischemic heart disease. In another aspect, the inflammation isassociated with brain death, preferably wherein levels of circulatingendogenous α-MSH or α-MSH in brain tissue is reduced. In yet anotheraspect, the therapeutic agent is used to treat a subject with an α-MSHor MC1-receptor mediated disorder or disease.

In another aspect, the present invention is directed towards treatmentof a subject with melanoma. In one aspect, the present inventionattenuates or ameliorates melanoma in subjects. In yet another aspect,the compounds of the present invention are used in assays for melanomadetection or imaging.

Kits Comprising the Disclosed Compounds

The invention also provides kits for carrying out the therapeuticregimens of the invention. Such kits comprise therapeutically effectiveamounts of a peptide having specific activity as MC1R modulators, inpharmaceutically acceptable form, alone or in combination with otheragents, in pharmaceutically acceptable form. Preferred pharmaceuticalforms include peptides in combination with sterile saline, dextrosesolution, buffered solution, or other pharmaceutically acceptablesterile fluid. Alternatively, the composition may be lyophilized ordesiccated. In this instance, the kit may further comprise apharmaceutically acceptable solution, preferably sterile, to form asolution for injection purposes. In another embodiment, the kit mayfurther comprise a needle or syringe, preferably packaged in sterileform, for injecting the composition. In other embodiments, the kitfurther comprises an instruction means for administering the compositionto a subject. The instruction means can be a written insert, an audiorecording, an audiovisual recording, or any other means of instructingthe administration of the composition to a subject. In one embodiment,the kit comprises (i) a first container containing a peptide havingMC1R-specific modulatory activity; and (ii) instruction means for use.

Additional Embodiments of Method of Treatment

In some embodiments, the invention provides a method to reduce orinhibit transplant rejection in a subject in need thereof comprisingadministering to said subject a pharmaceutical composition comprising apharmaceutically acceptable excipient and an effective amount of atleast one of the peptide compounds provided herein. In some embodiments,the invention provides a method to reduce or inhibit an immune responseof the subject elicited by transplanted tissue, cells or organ.Non-limiting examples of a transplanted tissue are an allograft inexperimental heart transplantation and pancreatic islet cells.

Immunosuppressive Activity in Experimental Autoimmune Encephalomyelitis(EAE) Model

Novel D-amino acid peptide analogs of α-MSH were generated and evaluatedfor immune modulatory effects in the experimental autoimmune uveitis(EAU) and experimental autoimmune encephalomyelitis (EAE) models as wellas in a lipopolysaccharide (LPS) surrogate inflammatory disease model.Treatment with RI α-MSH analog reduced clinical disease scores andincidence of disease in the EAU model and in a MOG induced EAE mousemodel. In addition, TNF-α and IL-10 mRNA expression levels in spleen andlymph node of MOG primed mice was decreased in treated mice. In a LPSinduced systemic inflammation model, α-MSH and analog treatmentdecreased serum cytokine levels. These data indicate that these novelα-MSH analogs have the potential for therapeutic use in inflammation andautoimmune disease.

Materials and Methods

Peptides.

α-MSH (SYSMEHFRWGKPV) (SEQ ID NO:8) was purchased from Bachem (King ofPrussia, Pa.). A D-amino acid RI α-MSH analog (vpkgwrfhemsys), alaninesubstituted peptide of RI α-MSH, (Stearyl) HfRW (820), (Ph(CH₂)₃CO) HfRW(819), and RI α-MSH analog 891 [vpkGwr(D-Cha)hsiiss] (SEQ ID NO:5) weresynthesized by Genzyme Corporation with a standard solid phasemethodology and was purified by reverse phase HPLC. A scrambled D aminoacid control peptide (ksrsmgvfpeyh) was synthesized by GenzymeCorporation. An irrelevant D amino acid control peptide (plykkiikklles)was synthesized by Genzyme Corporation.

Animals.

Five to six week old female B10.RIII-H2r mice were purchased fromJackson Laboratories (Bar Harbor, Me.).

Six to eight week old female or male C57BL/6 mice were purchased fromJackson Laboratories (Bar Harbor, Me.). All protocols for animal studiesmet with approval from the Institutional Animal Care and use Committee(IACUC) at Genzyme Corporation.

EAU Induction.

Female B10.RIII were injected subcutaneously at two sites (betweenshoulder blades and in pelvic region) with a total of 200 μginterphotoreceptor binding protein peptide (IRBP) 161-180 (New EnglandPeptide; Fitchburg, Mass.), respectively in 2 mg/ml complete Freund'sadjuvant (CFA; Sigma, St. Louis, Mo.) with mycobacterium tuberculosisH37Ra (Difco, Detroit, Mich.).

Eye Scoring.

The eyes of mice are dilated using one or two drops of Mydriacyl 1%(Alcon; Humacao, Puerto Rico) and rested in a darkened room forapproximately 5 min. Mice are manually restrained and the retinas ofboth eyes are visualized using an indirect ophthalmoscope with a 78diopter lens. The eyes are scored for inflammation using a progressivescoring system between 0-5. Score 0: Normal retina. Score 1: Vascularinflammation proximal to the optic nerve. Score 2: <5 inflammatorylesions confined to one quadrant of the eye. Score 3: >5 inflammatorylesions in more than one quadrant of the eye. Score 4: Inflammatorylesions are contiguous. Score 5: Retinal detachment. Whole eyes frommice were harvested and placed in PBS. The eyes were embedded in OCTmedia for frozen fixation. 5-μm sections were cut through thepapillary-optic nerve plane and stained with hematoxylin and eosin.

Binding Studies to Melanocortin Receptors.

Binding profiles for α-MSH, RI α-MSH, and scrambled peptide weremeasured to melanocortin receptors: 1, 3, 4, and 5. Binding was doneusing a competitive binding assay with (Nle4,D-Phe7) α-MSH (Bachem).Binding analysis was done by Cerep laboratories (Paris, France) withcoded samples.

Binding of the peptides was also followed by a competition with¹²⁵I-NDP-MSH for binding the melanocortin receptors 1, 3, 4, and 5 usingmembrane preparations from HEK293 cell lines. Peptides were mixed with¹²⁵I-(Nle4,D-Phe7)-α-MSH (PerkinElmer, Boston Mass.) in V-bottom 96 wellplates in binding buffer (25 mM HEPES pH7, 1.5 mM CaCl₂, 1 mM MgSO₄, 100mM NaCl, 0.2% BSA) and mixed with membrane preparations from HEK293transfected cell lines (Perkin Elmer, Boston Mass.) containing 1-10 fmolreceptor for 1 hr at 25° C. (Nle4,D-Phe7)-NDP-MSH (Bachem) at 3 μM wasused as a positive control. The mixtures were filtered through 96-wellGFC filters (Millipore, Billerica, Mass.), washed 3 times with bindingbuffer without BSA, dried and counted.

cAMP Measurement.

B16-F1 (melanoma cell line) cells from American Type Culture Collection(Manassas, Va.) were seeded into flat bottom 96 well plates at5×10⁴/well and cultured overnight in DMEM (Cambrex, Walkersville, Md.)supplemented with 2 mM glutamine, antibiotics (100 U/ml penicillin and100 U/ml streptomycin) and 10% heat-inactivated fetal bovine serum(Invitrogen, Grand Island, N.Y.). Cells were then treated with nativeα-MSH (0.01-1000 ng/ml), retro-inverso α-MSH (0.01-1000 ng/ml), or ascrambled D amino acid control peptide (0.01-1000 ng/ml) for 30 min.Cells were lysed and intracellular cAMP levels were measured by anenzyme immunoassay kit (Amersham Biosciences, Piscataway, N.J.).Forskolin (Sigma, St. Louis, Mo.) at 100 μM served as positive controls.

EAE Induction and Scoring.

Female C57BL/6 mice were immunized with an emulsion of MOG35-55 peptide(200 μg/mouse; New England Peptide; Fitchburg, Mass.) in completeFreund's adjuvant (CFA; Sigma, St. Louis, Mo.) containing 0.6 mgMycobacterium tuberculosis (Difco; Detroit, Mich.). The emulsion wasdelivered in a volume of 0.2 ml per mouse by subcutaneous injection totwo sites. Bordetella pertussis toxin (PTX; Sigma) in PBS was used at adose of 400 ng/animal via an i.p. administration route and isadministered on day 0 and day 3. Mice were monitored daily for paralyticsymptoms of EAE. The mice were scored for clinical symptoms using aprogressive scoring system between 0-5. Score 0: no disease; Score 1:flaccid tail; Score 2; hind limb weakness; Score 3: hind limb paralysis;Score 4: Front limb weakness/partial paralysis; Score 5; death. Spinalcord and brain were harvested and embedded in paraffin for hematoxylinand eosin staining.

Proliferation Assay.

C57BL/6 mouse splenocytes and lymph node cells were cultured in 96 wellplates 5×105 cells/well. MOG35-55 or OVA 257-264 peptide was added towells at a concentration of 25 μg/ml. Supernatant was collected at 48hrs after MOG peptide stimulation. After 3 days in culture cells werepulsed with [3H] thymidine at 1 μCi/well for 8 hrs. Thymidineincorporation was measured by cpm.

Cytokine RNA Analyses-RT-QPCR.

Total RNA was extracted from tissues with TRIZOL® (Invitrogen, Carlsbad,Calif.). 1 μg of total RNA was reverse transcribed and used inquantitative PCR by using SYBR® green incorporation with reagents fromApplied Biosystems (Foster City, Calif.) on an ABI 7900. A cDNA standardwas run in each PCR for cytokine targets and message concentrations werenormalized to mouse β-actin. Primers used to obtain these data were asfollows: mouse TNFα forward-ggcaggtctactttggagtcattgc (SEQ ID NO:27) andreverse-acattcgaggctccagtgaattcgg (SEQ ID NO:28) (1). The sequences formouse IL-10 primers were forward: tgctatgctgcctgctctta (SEQ ID NO:29)and reverse: tcatttccgataaggcttgg (SEQ ID NO:30) (2). The sequences formouse β-actin primers were: forward gtgggccgctctaggcaccaa (SEQ ID NO:31)and reverse ctctttgatgtcacgcacgatttc (SEQ ID NO:32) (3).

CBA Analysis.

Flow cytometric analysis of inflammatory cytokine profiles of mouseserum or cell supernatant were measured using the Cytometric Bead ArrayKit (CBA) made by BD Biosciences (San Jose, Calif.). Samples wereprocessed according to the manufacturer's protocol.

mMC1R RNA Analyses-RT-QPCR.

Total RNA was extracted from tissues with TRIZOL® (Invitrogen, Carlsbad,Calif.). 1 μg of total RNA was reverse transcribed and used inquantitative PCR by using a TAQMAN® kit with reagents from AppliedBiosystems (Foster City, Calif.) on an ABI 7900. A cDNA standard was runin each PCR for mMC1R and message concentrations were normalized toEukaryotic 18S endogenous control (VIC/MGB probe, Applied Biosystemspart #4319413E). Primers for mMC1R were as follows: forwardCTCTGCCTCGTCACTTTCTTTCTA (SEQ ID NO:24) and reverseAACATGTGGGCATACAGAATCG (SEQ ID NO:25) and probe CCATGCTGGCACTCA (SEQ IDNO:26). These were designed using PRIMER EXPRESS® 3.0 (AppliedBiosystems, Foster City, Calif.).

LPS Model.

Male C57BL/6 mice were challenged with LPS (1 ug/mouse), i.p. After 30min mice were treated with dexamethasone (2 mg/kg; Sigma), RI-α-MSHanalog 891, or RI-α-MSH. At 2 hrs after LPS challenge, mice were bledfor serum. Cytokine analysis was measured by flow cytometry with acytometric bead array kit (BD Biosciences).

Statistical Analysis.

The results are expressed as the mean±SD. Each experiment was repeatedat least twice. To analyze the results a one-way ANOVA and the Tukeymultiple comparison test was used.

The following examples are offered to illustrate but not to limit theinvention.

Example 1 Immunosuppressive Activity in Experimental Autoimmune Uveitis(EAU) Model

Autoimmune uveitis is an inflammatory disorder of the eye that can leadto pain and vision loss. Steroids and immunosuppressive drugs arecurrently the only therapeutics for uveitis and often have seriousocular and systemic toxicities. Therefore, safer alternativetherapeutics are desired.

EAU is an animal model of human uveitis which affects 2.3 millionindividuals in the United States. Immunization with interphotoreceptorretinoid binding protein (IRBP) or its peptide fragments or retinalS-antigen (S-Ag) with adjuvant can induce disease in susceptible strainsof rodents. The disease involves infiltration of inflammatory cells inthe retina of the eye and photoreceptor damage with natural recoverywithout spontaneous relapse. The adoptive transfer of uveitogenic Tcells in syngeneic rodent recipients suggests that uveitis is an organspecific T cell mediated autoimmune disease like many other similarautoimmune diseases such as multiple sclerosis, type 1 diabetes andrheumatoid arthritis.

The ocular microenvironment is an immune privileged site in whichmechanisms to maintain immunosuppression are in place to prevent localinflammation. Several neuropeptides that are expressed by neurons in theocular tissue help to sustain immune privilege in the eye. One of theseneuropeptides is α-MSH which is constitutively expressed in the ocularmicroenvironment at a physiological concentration of 30 pg/ml.

Systemic administration of native α-MSH during the induction ofendotoxin-induced uveitis in rats inhibited the number of infiltratingcells and IL-6, TNF-α, MCP-1, MIP-2, and nitric oxide levels in aqueoushumor in a dose dependent manner. In a mouse EAU model administration ofα-MSH at the peak time of retinal inflammation suppressed the severityof disease. α-MSH can induce T regulatory cells through the melanocortin5 receptor (MCR-5) on T cells and suppress EAU.

The efficacy of native α-MSH treatment in the murine posterior uveitismodel was evaluated. Uveitis was induced in B10.RIII mice with aninjection of IRBP 161-180 and CFA. Onset of disease occurred around day10 after priming. When eye scores of 2-3 were reached on day 13, micewere administered native α-MSH at 200 μg/mouse, i.v. for 7 consecutivedays or left untreated. Since prophylactic treatment may targetactivities in the priming phase of the disease rather than the effectorphase, treatment was initiated when active retinal inflammation wasobserved. In addition, this treatment strategy better recapitulatesclinical application. Mice treated with the α-MSH showed a reduction ofmean clinical eye scores throughout the 7 day course of treatmentcompared with untreated mice (FIG. 1a ). α-MSH treated mice showed amaximum mean eye score of 2.83±0.39 on day 13 in comparison to theuntreated group of mice which reached a maximum mean eye score of3.67±0.52 on day 15.

Example 2 Comparison of Native α-MSH to Dexamethasone in the B10.RIIIExperimental Autoimmune Uveitis Model

Current forms of therapy for uveitis include corticosteroids andimmunosuppressive agents. The efficacy of native α-MSH was compared to aknown corticosteroid therapy, dexamethasone. Uveitis was induced inB10.RIII mice with IRBP161-180 and CFA injections. At time of diseaseonset, when mice reached a clinical eye score of 1, mice wereadministered daily intraperitoneal injections of native α-MSH (100μg/mouse), 0.2 mg/kg of dexamethasone, or 2.0 mg/kg of dexamethasone.Mice were treated daily for 21 days. Mice treated with native α-MSHshowed a significant reduction of mean clinical eye scores throughoutthe course of treatment compared with PBS control mice (FIG. 1B). Datashowed that daily i.p. administration of native α-MSH suppressed uveitisto a greater degree than the dexamethasone treatment at either the 0.2mg/kg or 2.0 mg/kg dose.

Example 3 A Novel Retro-Inverso α-MSH Analog Specifically Binds to MCR-1

A novel, stable D-amino acid peptide analog of native α-MSH wassynthesized (RI α-MSH) and evaluated for immune modulatory capabilitiesin vitro and in the experimental autoimmune uveitis (EAU) model. Bindingstudies indicated that unlike native α-MSH, RI α-MSH binds specificallyto the anti-inflammatory α-MSH receptor (MCR-1) but none of the otherα-MSH receptors (MCR-3, 4, or 5).

The binding of the RI α-MSH analog to melanocortin receptors (MCR) wasanalyzed. A competitive binding assay using a MCR panel including MCR 1,3, 4, and 5 was performed. Binding of the peptides was followed by acompetition with ¹²⁵I-NDP-MSH as described previously. Native α-MSHbound to MCR 1, 3, 4, and 5. However, unlike native α-MSH, RI α-MSH wasfound to bind only to MCR-1 which regulates inflammatory responses(Table 1). A scrambled D amino acid control peptide did not bind to anyof the melanocortin receptors. The development of an α-MSH analog withspecific binding to MCR-1, and the exclusion of MCR-3 and MCR-4 binding,can decrease potential side-effects that native α-MSH produces throughbinding these receptors.

TABLE 1 Ki (nM, except as noted) No. N-terminus Sequence C-terminus MC1RMC3R MC4R MC5R MSH Ac SYSMEHFRWGKPV Amide  0.41 ± 0.14   23  41 ~1500(SEQ ID NO: 8) RI-MSH Ac vpkGwrfhemsys Amide  4.6 ± 1.3 >30 μM  >30 μM ~25 μM (720e) (SEQ ID NO: 7) 804 Ac vpkGwrfhemsya Amide nd(SEQ ID NO: 34) 805 Ac vpkGwrfhemsas Amide (SEQ ID NO: 35) 806 AcvpkGwrfhemays Amide (SEQ ID NO: 36) 807 Ac vpkGwrfheasys Amide 1400 ±190 (SEQ ID NO: 37) 808 Ac vpkGwrfhamsys Amide 2.5 (SEQ ID NO: 38) 809Ac vpkGwrfaemsys Amide 1200 ± 270 (SEQ ID NO: 39) 810 Ac vpkGwrahemsysAmide    >30 μM (SEQ ID NO: 40) 811 Ac vpkGwafhemsys Amide 13 ± 7 μM(SEQ ID NO: 41) 812 Ac vpkGarfhemsys Amide      3 μM (SEQ ID NO: 42) 813Ac Vpkawrfhemsys Amide (SEQ ID NO: 43) 814 Ac vpaGwrfhemsys Amide(SEQ ID NO: 44) 815 Ac vakGwrfhemsys Amide (SEQ ID NO: 45) 816 AcapkGwrfhemsys Amide (SEQ ID NO: 46) 817 Ac aaaGwrfhemsys Amide(SEQ ID NO: 47) 818 Ac kkkGwrfhemsys Amide (SEQ ID NO: 48) 819(Ph(CH₂)₃CO) HfRW Amide 0.07  277  27 ~3000 (SEQ ID NO: 49) 820 StearylHfRW Amide 1.3 1371 390   860 (SEQ ID NO: 49) 847 H₂N vpkGwrfh(CH₂)₃Phenyl) 104 ± 72 (SEQ ID NO: 50) 847int H₂N vpkGwrfh OH 150(SEQ ID NO: 50) 857 Ac vp(D-Ornithine)Gwrfhemsys Amide 3.0(SEQ ID NO: 51) 858 Ac vp(D-Norleucine)Gwrfhemsys Amide 18(SEQ ID NO: 52) 859 Ac vpkG(3-benzothienyl-D- Amide 206 Alanine)rfhemsys(SEQ ID NO: 53) 860 Ac vpkG(5-hydroxy-D- Amide 96 Trp)rfhemsys(SEQ ID NO: 54) 861 Ac vpkG(5-methoxy-D- Amide 75 Trp)rfhemsys(SEQ ID NO: 55) 862-b Ac vpkGfrfhemsys Amide 128 (SEQ ID NO: 56) 863 AcvpkGwqfhemsys Amide   >100 μM (SEQ ID NO: 57) 864 Ac vpkGwnfhemsys Amide   >30 μM (SEQ ID NO: 58) 865 Ac vpkGwhfhemsys Amide 2300(SEQ ID NO: 59) 866 Ac vpkGwr(4-fluoro-D- Amide    >30 μMphenylglycine)hemsys (SEQ ID NO: 60) 867 Ac vpkGwr(3-pyridyl-D- Amide480 Alanine)hemsys (SEQ ID NO: 61) 868 Ac vpkGwr(2-thienyl-D- Amide 5.4Alanine)hemsys (SEQ ID NO: 62) 869 Ac vpkGwr(D-Cha)hemsys Amide   2.2 ±0.44 >30 μM  >10 μM  ~25 μM (SEQ ID NO: 63) 870 Ac vpkGwrwhemsys Amide  ~2.5 μM (SEQ ID NO: 64) 871 Ac vpkGwr(4-Nitro-D- Amide 324 Phe)hemsys(SEQ ID NO: 65) 872 Ac vpkGwrfremsys Amide 13.5 (SEQ ID NO: 66) 873 AcvpkGwrfwemsys Amide 1900 (SEQ ID NO: 67) 874 Ac vpkGwrffemsys Amide  >100 μM (SEQ ID NO: 68) 875 Ac vpkGwrfhdmsys Amide 400 (SEQ ID NO: 69)876 Ac vpkGwrfh(D-Citrulline)msys Amide nd (SEQ ID NO: 70) 877 AcvpkGwrfhe(α-methyl-D- Amide 282 Met)sys (SEQ ID NO: 71) 878 AcvpkGwrfhe(D-buthionine)sys Amide  2.3 ± 0.8 ~40 μM  >30 μM  ~30 μM(SEQ ID NO: 72) 879 Ac vpkGwrfheksys Amide 832 (SEQ ID NO: 73) 880 AcvpkGwrFhsiiss Amide  1.8 ± 3.5 ~18 μM  ~10 μM  >30 μM (SEQ ID NO: 4) 881Ac wrFh C₃-Phenyl  >10 μM (SEQ ID NO: 74) 882 Ac wrFh (1,6-diamino- 120(SEQ ID NO: 74) hexane)stearyl 883 Ac wrFh Amide   >100 μM(SEQ ID NO: 74) 884 Ac vpkGwrFhemsys Amide 72 >30 μM  ~30 μM  >30 μM(SEQ ID NO: 75) 886 Ac vpkgwrfhsiiss Amide   1.0 ± 0.43 ~19 μM  ~10 μM ~20 μM (SEQ ID NO: 76) 890 Ac vpkGwr(D-Cha)he(d- Amide   1.9 ± 0.01~15 μM  ~14 μM ~4.5 μM Buthionine)sys (SEQ ID NO: 77) 891 AcvpkGwr(D-Cha)hsiiss Amide  0.43 ± 0.01  ~8 μM ~3.2 μM ~2.5 μM(SEQ ID NO: 5) 892 Ac SYSMEH(L-Cha)RWGKPV Amide 0.51 (SEQ ID NO: 6) 893Ac vpkGWrfhemsys Amide 6.5 ~28 μM  >30 μM  ~27 μM (SEQ ID NO: 78) 894 AcvpkGwRfhemsys Amide 380 >30 μM  >30 μM  >30 μM (SEQ ID NO: 79) 895 AcvpkGwrfHemsys Amide 19 ~37 μM  ~11 μM  ~37 μM (SEQ ID NO: 80) Lower caseletters represent D-isomer amino acids; bold characters representchanges from RI α-MSH.

Binding of the peptides was followed by a competition with ¹²⁵I-NDP-MSHfor binding the melanocortin receptors 1, 3, 4, and 5 using membranepreparations from HEK293 cell lines. As shown in FIG. 18, RI α-MSHshowed a very strong selectivity for MC1R, with Ki values in excess of30 μM at both MC 3, 4 and 5R, whereas α-MSH showed significant bindingto all four receptors, with less than 100-fold selectivity for MC1Rrelative to MC3R.

Example 4 Treatment with Retro-Inverso α-MSH Analog Ameliorates Diseasein EAU

Immunomodulatory effects of the novel retro-inverso α-MSH peptide analogwas observed in the experimental autoimmune uveitis mouse (EAU) modeland compared results with the native α-MSH peptide. Systemic delivery ofRI α-MSH at onset of disease or during late stage disease dramaticallyand reproducibly ameliorated uveitis. In addition, treatment with thenovel RI α-MSH peptide analog suppressed uveitis with a similarmagnitude to the native α-MSH peptide. These data indicate that thenovel RI α-MSH analog shows anti-inflammatory activities and hastherapeutic use in uveitis and other autoimmune diseases andinflammation.

EAU was induced in female B10.RIII mice with IRBP 161-180. The efficacyof the RI α-MSH and native α-MSH as a therapeutic rather than aprophylactic treatment was examined. Mice were treated daily byintravenous injection with 100 μg/mouse RI α-MSH, 100 μg/mouse nativeα-MSH, or PBS beginning when mice reached a moderate disease stage ofuveitis (eye scores of 2). As seen in FIG. 2A, control PBS-treated micereached maximum eye scores on day 16 with a mean eye score of 2.75±0.68.However, mice treated daily with the RI α-MSH analog or native α-MSHshowed a significant reduction of mean clinical eye scores throughoutthe course of treatment compared with PBS control mice. On day 16, fourdays after the start of treatment, 5 of the 8 mice in the PBS treatedgroup had a maximum eye score of 3 or greater whereas only 1 of the 8native α-MSH treated mice and 0 of the 8 RI α-MSH treated mice had amaximum eye score of 3 or greater (FIG. 2B).

Example 5 Retinal Images and Histological Examination of SubjectsTreated with RI α-MSH Show Suppression of Disease in EAU Models

Retinal images from mice treated daily with RI α-MSH or native α-MSHbeginning at late stage uveitis show suppression of disease comparedwith PBS control mice. Fundoscopic images were acquired on day 13 afterstart of treatment (FIG. 3). Disease in the PBS treated group stabilizedor advanced and had a median eye score of 3 with severe vasculitis andinflammatory lesions throughout the eye (FIG. 3A). However, diseaserapidly resolved in both the RI α-MSH and native α-MSH peptide treatedmice. Retina images showed eye scores of 1 with inflammation only at theoptic nerve (FIGS. 3B and 3C). Maximum eye scores for individual mice ineach group are shown in FIG. 3D. In the PBS treated group 7 of the 11mice had eye scores 3 or greater. However, only 2 of the 11 mice in thenative α-MSH and RI α-MSH had eye scores of 3 or greater.

Histological examination of the eyes 10 days after the start of dailytreatment showed that both RI α-MSH and native α-MSH reduced pathologyin the eye (FIG. 4). The native α-MSH and RI α-MSH treated mice shownormal retinal architecture with slight inflammation in the optic nerveregion (FIGS. 4B and 4C). In contrast, PBS treated mice show ocularinflammation and tissue damage Inflammation is seen in the retina andoptic nerve regions with photoreceptor damage (FIG. 4A).

Example 6 Comparison of Intraperitoneal Administrations of Retro-Inversoα-MSH to a Scrambled Control Peptide in EAU

Uveitis was induced in B10.RIII mice with IRBP161-180 and CFAinjections. We evaluated initiating treatment during late-stage disease(eye scores of 2-3) with RI α-MSH by daily intraperitoneal injectionsand compared efficacy with a control scrambled D amino acid peptide.Mice were treated daily, i.p., for 13 days with RI α-MSH or scrambledpeptide at 100 μg/mouse. Mice treated with RI α-MSH showed a significantreduction (p<0.04) in the mean clinical eye scores on days 15, 21 and 23of the disease course compared with scrambled peptide control mice (FIG.5). Individual maximum eye scores on day 23 after disease inductionshowed 75% of mice with scores ≤1 in the RI α-MSH compared with none ofthe mice in the control scrambled peptide group with scores ≤1.Individual weights of the mice were recorded at 4 timepoints throughoutthe course of disease. All mice in both the RI α-MSH treated andscrambled peptide treated groups maintained their weight or showednormal weight gain (data not shown). Daily intraperitoneal dosing withthe peptides did not result in weight loss. In addition theintraperitoneal route of administration of native α-MSH or RI α-MSH at100 μg/mouse had efficacy in uveitis when treatment was administered atonset of disease, when mice reach a clinical eye score of 1 (data notshown). Therefore, intraperitoneal route of administration of RI α-MSHshowed significant reduction in disease scores compared with a scrambledcontrol peptide.

Example 7 Administration of RI α-MSH at Various Dosages to EAU Model

We also examined the efficacy and optimal dosing of retro-inverso α-MSHtreatment during late-stage severe uveitis. B10.RIII mice were injectedwith IRBP161-180 and CFA to induce uveitis. Mice were treated daily,i.p., with PBS, control scrambled peptide (100 μg/mouse), or RI α-MSH at100 μg, 10 μg or 3 μg/mouse. Treatment was initiated at peak time ofdisease when mice reached eye scores of 4 which included inflammatorylesions throughout the posterior segment of the eye and possiblehemorrhaging. Disease in PBS and scrambled peptide control groupsremained severe (FIG. 6). In contrast, treatment with RI α-MSH at 100 μgor 10 μg/mouse rapidly ameliorated uveitis (p≤0.05). In addition, dosesof 10 and 100 μg/mouse were equally effective. The 3 μg/mouse dose of RIα-MSH did not reduce or inhibit disease.

Example 8 Effect of Retro-Inverso α-MSH on cAMP Levels

cAMP levels in B16-F1 melanoma cells after treatment with native α-MSH,RI α-MSH, or a scrambled control peptide were examined. The B16-F10melanoma cell line expresses a high number of MCR1 receptors (3000-4000receptors/cell) compared with macrophages cell lines which express only100-200 receptors/cell. Therefore we selected to examine the effect ofthe RI α-MSH treatment on the melanoma cell line. Murine melanoma B16-F1cells were treated with native α-MSH, RI α-MSH, or control scrambledpeptide at concentrations 1 pg/ml-1 μg/ml. After 30 min, cells werelysed and intracellular cAMP was measured by an enzyme immunoassay.Forskolin, commonly used to raise cAMP levels, control (100 μM)treatment of cells showed an increase in cAMP (3455.39±406.6 SD). cAMPlevels were significantly elevated in a dose dependent manner in cellstreated with native α-MSH compared with untreated cells (FIG. 7A). TheRI α-MSH analog also significantly increased cAMP levels in a dosedependent manner compared with untreated or scrambled peptide control.However, a higher concentration of RI α-MSH (100 ng/ml) was necessary toincrease cAMP compared with native α-MSH which increased cAMP at a 10pg/ml concentration. This difference in concentration of peptidenecessary to increase cAMP levels may be the result of the bindingaffinity to the MCR1 receptor.

Example 9 Sequence Variation and Effects on cAMP Levels and Binding MC1R

MSH binds to MC1R, MC3R, MC4R, and MC5R, with MC1R being one of thedesired targets for immune mediated diseases. Retro-inverso MSH (RI-MSH)has been engineered to have enhanced plasma stability (FIG. 17) and MC1Rselectivity, but its affinity for MC1R is 11-fold lower than that of thenative MSH peptide (Table 1). To restore MC1R affinity, we grafted intoRI-MSH modifications known to improve MC1R affinity of MSH. Of the threereplacements of the N-terminal SYSME sequence—fatty acyl, phenyl butyricacid, and SSIIS sequence—that enhance MC1R affinity of MSH, only thelast one led to significant improvements for RI-MSH. D-alanine scanninganaloging of RI-MSH exhibited a similar structure activity relationshipas alanine scanning analoging of MSH, but stereochemistry inversionscanning of the core 4-residue MC1R binding region suggests significantdifferences between MSH and RI-MSH. Furthermore, cyclohexylalaninesubstitution at the key phenylalanine residue improved RI-MSH, but notMSH binding to MC1R. Combining cyclohexylalanine and SSIIS substitutionsled to full restoration of MSH affinity for MC1R, while retaining theretro-inversion configuration critical for the improved stability andthe high MC1R selectivity of RI-MSH.

A set of alanine scanning analogs (peptides 804-816) of retro-inversoMSH (RI-MSH) were prepared and tested for cAMP induction in B16/F1murine and M624 human melanoma cells. A subset based on the cAMP resultswere then tested for binding MC1R. The observed Ki values for bindingMC1R are shown in Table 1.

Murine melanoma B16-F1 cells were treated with native α-MSH, RI α-MSH,scrambled peptide control, KPV, or alanine substituted peptides of RIα-MSH at 1 μg/ml (FIG. 7B). Forskolin control (100 μM) treatment ofcells showed increase in cAMP (3294.82±54.53). cAMP levels weresignificantly elevated in cells treated with native α-MSH and RI α-MSHcompared with untreated, scrambled peptide, or KPV treated cells.Alanine substituted peptides designated 810, 811, and 812 showed noincrease in cAMP activity and exhibited levels of cAMP equivalent tountreated, scrambled peptide or KPV treated cells. Peptides designated810, 811, and 812 have alanine substitutions in the central coretetrapeptide sequence (D-Trp D-Arg D-Phe D-His region; AA 5-8) (variantdisclosed in SEQ ID NO:2) proposed to be involved in native α-MSHbinding to the melanocortin receptor and its biological activity.Alanine substitutions at the N terminal or C terminal regions of the RIα-MSH peptide did not affect cAMP accumulation in the melanoma cells.Alanine substitutions in the methionine and histidine amino acids (807and 809) also showed a reduction in cAMP accumulation although not asgreat as that seen in the core tetrapeptide sequence (810, 811 or 812).The residues in the core tetrapeptide (wrfh) (variant disclosed in SEQID NO:2) and to a lesser extent, the methionine 4 of RI-MSH are criticalfor binding to MC1R.

Another means shown to increase the affinity of MSH for MC1R has beendemonstrated using selections based on phage display of MSH variantsequences. A highly MC1R-selective sequence (MS05) was subsequentlyfound by recombination of the phage display-selected peptide and aportion of the parent MSH sequence, which yielded a subnanomolaraffinity for MC1R. Unexpectedly, the retro-inverso version of thissequence showed a higher affinity for MC1R (1 nM, Table 1 peptide 886)than RI-MSH (4 nM), even though MS05 was reported to have a slightlylower affinity for MC1R than MSH (Ki 0.865 nM vs. 0.557 nM for MSH).

Substitution of a number of non-natural amino acid residuesincorporating slight differences in charge and structure at each of thepositions of RI-MSH showed that MC1R tolerates only highly conservativechanges. All but 3 showed lesser or equivalent binding. Two changesprovided a significant increase in affinity for MC1R: substitution ofthe D-phenylalanine by D-cyclohexylalanine (D-Cha, peptide 869; 2.2 nMKi) and substitution of the D-methionine by D-buthionine (peptide 878,2.3 nM Ki). Substitution with L-cyclohexylalanine was performed and wasfound to slightly inhibit binding to MC1R (peptide 892, Ki of 0.51 nMvs. 0.41 nM for MSH). Unexpectedly, combination of the buthionine andcyclohexylalanine substitutions failed to produce a further significantincrease in affinity for MC1R (peptide 890, 1.9 nM Ki). However,combination of the change comprising substitution of the retro-inversoN-terminal sequence of MS05 (siiss) (SEQ ID NO:3) for the C-terminalsequence (emsys) (SEQ ID NO:34) and D-cyclohexylalanine for D-Phe inRI-MSH produced a greater enhancement of binding than each of thechanges alone, indicating the effects are synergistic. The resultingpeptide (891) showed a Ki for MC1R indistinguishable from MSH. Althoughthis and the other changes also produced increases in the affinity forthe other MCR's, the Ki values were still in the micromolar range,indicating the selectivity of RI-MSH was largely preserved. The changesare illustrated in FIGS. 20A-C. A representative competitive bindingassay is shown in FIG. 21. See Table 1 for the observed Ki values.

Example 10 Treatment with RI α-MSH Reduces Clinical Disease Scores inEAE

The effect of administration of native α-MSH and RI α-MSH analog in achronic progressive EAE mouse model was evaluated. Female C57BL/6 micewere immunized with 200 μg MOG 35-55 peptide emulsified with CFA.Pertussis toxin was administered on day 0 and 2. Mice were monitoreddaily for signs of clinical symptoms and weight loss. Mice wereevaluated for symptoms of paralysis starting on day 8 and were scored ona grading system from 0-5. The appearance of clinical symptoms ofparalysis manifested around day 9-11 in most mice (FIG. 8A). Dailyintraperitoneal (i.p.) treatment with α-MSH or RI α-MSH peptide at 100μg/mouse or PBS control began on day 10. Mice treated with 100 μg of RIα-MSH showed a significant reduction in the mean clinical disease scorecompared with PBS control (FIG. 8A). Significance of p≤0.05 was reachedon days 14-22 in the RI α-MSH compared with PBS vehicle control. Howeverthe native α-MSH peptide treatment did not have an effect on diseaseinduction or progression. The maximum percent incidence of disease inthe RI α-MSH treated group of mice (20%) was also reduced compared withthe PBS (80%) or native α-MSH treated (75%) groups of mice.

Example 11 Dosage Variation of RI α-MSH in EAE

The therapeutic effect of RI α-MSH was apparent at the 100 μg/mousedose. Treatment with α-MSH or RI α-MSH peptide at 100 μg/mouse and 30μg/mouse was tested in the MOG EAE mouse model. Daily i.p. treatmentbegan on Day 10 after MOG immunization. Daily dexamethasone (2 mg/kg)treatment was added as a control therapeutic. Mice that were treatedwith 100 μg of RI α-MSH repeatedly showed a reduction in the meanclinical disease score compared with PBS control (FIG. 9). However the30 μg/mouse dose of RI α-MSH did not have a significant effect onreducing mean clinical scores. Dexamethasone treatment also showed areduction in disease scores throughout the course of disease. Althoughmice treated with native α-MSH had a lower mean clinical score than PBS,the α-MSH treated mice did not show similar efficacy as the RI α-MSH ordexamethasone treated mice. The percent incidence of disease in the RIα-MSH treated group reached a maximum of 35% and the dexamethasone 40%compared with PBS treated mice which showed a 75% maximum incidence ofdisease (FIG. 9). These data indicate that treatment with the RI α-MSHpeptide analog in EAE significantly decreases mean clinical diseasescores and incidence of disease.

Example 12 CNS Histology

Spinal cords were harvested on day 24 after disease induction in PBS andRI α-MSH treated mice. Hematoxylin and eosin staining of spinal cordsections were used to assess the degree of inflammation and number oflesions. The pathology of EAE shows focal areas of infiltration ofinflammatory cells and demyelination. Histopathological evaluation ofthe spinal cord demonstrated efficacy of RI α-MSH treatment in the MOGEAE model. Slices from representative mice of the mean clinical scorefor each treatment group are shown in FIGS. 10A-10D. Data show extensiveinflammatory infiltrates in the PBS treated group of mice compared withRI α-MSH treated mice which lack focal area of inflammation.

Example 13 Measurement of TNF-α and IL-10 in the Spleen During DiseaseProgression

The mechanism of action of the effects of RI α-MSH treatment in EAE wasexamined. Native α-MSH has been reported to have an effect onmonocytes/macrophages by decreasing TNF-α levels and increasing IL-10.TNF-α and IL-10 mRNA levels in the spleen of MOG primed mice treatedwith RI α-MSH or native α-MSH were evaluated by quantitative PCR. Micewere immunized with MOG p35-55 and daily treatment with RI α-MSH ornative α-MSH started on Day 10. Spleen samples were harvested from miceon Days 1, 4 and 7 after start of daily treatment. Data show treatmentwith RI α-MSH and α-MSH significantly decreased (p≤0.001) TNF-α comparedwith PBS control after 7 days of daily treatment (FIG. 11E). Howeverthere was no significant change in the TNF-α level at previous timepoints (Day 1 and Day 4; FIGS. 11A and 11C). IL-10 mRNA levels were alsoreduced at the Day 7 timepoint in both the RI α-MSH and αMSH treatmentgroups compared with PBS control (FIG. 11F). However, no difference inIL-10 mRNA levels was detected at the earlier timepoints (FIGS. 11B and11D). Although native α-MSH treatment did not reduce mean clinicaldisease scores or percent incidence of disease, in this study α-MSHtreatment reduced both TNF-α and IL-10 mRNA levels in the spleencompared with PBS control by Day 17 of disease progression. RI α-MSHanalog is able to reduce TNF-α mRNA levels in the spleen after dailytreatment.

Example 14 Effect of RI α-MSH on Recall Response to MOG Peptide

The effects of treating mice with α-MSH or RI α-MSH in vivo on recallresponses to the MOG 35-55 peptide were evaluated. Mice were primed withMOG35-55 peptide and on Days 2-8 mice were either treated with PBS, 100μg α-MSH or 100 μg RI α-MSH. On Day 9, spleen and draining lymph nodeswere harvested and analyzed for recall responses to MOG35-55 peptide invitro by [3H] thymidine incorporation. Data show a significant decreasein proliferative responses of spleen cell to MOG35-55 peptide in theα-MSH group of mice compared with the PBS treated group (FIG. 12A). TheRI α-MSH treated group showed a slight reduction in recall responses toMOG35-55 peptide. However, recall responses to MOG peptide in the lymphnode cell population did not show a significant difference inresponsiveness between the PBS, α-MSH, and RI α-MSH treated groups (FIG.12B).

Cell supernatant was collected from the spleen cells that werestimulated with MOG35-55 peptide in vitro from each of the in vivotreatment groups (naïve, PBS, α-MSH, RI α-MSH). Cytokine levels wereevaluated through a cytometric bead array by flow cytometry. Data show adecrease in TNF-α, IFNγ, IL-6 and MCP-1 levels in both the α-MSH and RIα-MSH treated groups compared with the PBS treated group (FIGS. 12C and12D). The cytokine levels of spleen cells supernatant in both the α-MSHand RI α-MSH treated groups were similar to cytokine levels from spleencells of unprimed mice.

Therefore, RI α-MSH peptide treatment had an effect on cytokine recallresponses to MOG peptide but did not affect T cell proliferationresponses.

Example 15 Effects of Treating Mice with α-MSH or RI α-MSH During thePriming Phase of EAE

Mice were immunized with MOG 35-55 peptide and on days 2-8 treated dailywith PBS vehicle control, α-MSH, or RI α-MSH. Spleens from individualmice were harvested on Day 9 and mRNA cytokine expression wasquantitated using real time PCR. FIG. 13A-13D shows TNF-α and IL-10 mRNAexpression levels in the spleen of PBS, α-MSH or RI α-MSH treated micein the priming phase of disease. Naïve mice that were not immunized withMOG peptide were used to quantitate baseline mRNA levels of TNF-α andIL-10. Treatment with either α-MSH or RI α-MSH decreased levels of IL-10and TNF-α mRNA compared with PBS vehicle control.

Blood serum samples were also collected on day 9 of the study andevaluated for cytokine levels: TNF-α, MCP-1, IL-12, IL-10, and IL-6(FIGS. 13C and 13D). Data show a decrease in MCP-1 and IL-6 in both theα-MSH and RI α-MSH treated groups compared with the PBS control group.MCP-1 was significantly decreased (p<0.01) in the RI α-MSH treated groupcompared with PBS control. Additionally, the RI α-MSH treated groupshowed a decrease in TNF-α and IL-12 compared with the PBS group. Therewas no difference in serum IL-10 levels between the three treatedgroups.

Example 16 RI α-MSH does not have an Effect on Macrophage Markers

Splenic CD11b⁺ and F4/80⁺ macrophages were examined for surfaceexpression levels of CD86, CD40, and CD14 by flow cytometry after 7 daysof daily dosing with α-MSH or RI α-MSH in MOG 35-55 immunized mice. Datashow no difference in the expression levels of CD86, CD40 or CD14 in thesplenic CD11b⁺ or F4/80⁺ macrophage cell population in mice treated withα-MSH or RI α-MSH (FIGS. 14A and 14B). Results showed a similar findingwhen blood monocytes were examined for CD86, CD40, and CD14 expressionlevels (data not shown).

Example 17 Effects in the LPS Inflammation Mouse Model

Native α-MSH has been reported to inhibit inflammation by downregulatingTNF-α production through stimulation of melanocortin receptors (MCR), inparticular the melanocortin 1 receptor. LPS stimulates inflammatorymediators such as TNF-α, MCP-1, IL-6 and IFNγ and has been used as anacute inflammation model. Whether an α-MSH analog can suppressinflammatory cytokines in a LPS mouse inflammation model was examined.The levels of MC1R expression in the spleen and peritoneal macrophagesafter LPS administration were determined. C57BL/6 mice were injectedwith LPS, i.p. and at several timepoints post-LPS injection spleen andperitoneal macrophages were harvested for analysis. Data show nodiscernable difference in the levels of MC1R mRNA expression among anyof the timepoints in peritoneal macrophages (FIG. 15A). However, LPSadministration did increase mRNA levels of MC1R above naïve mice thatdid not receive a LPS challenge. In the spleen the levels of MC1R mRNAwere elevated 30 minutes post-LPS injection and then decreased by onehour post-injection (FIG. 15B). LPS similarly increased mRNA levels ofMC1R in the spleen above baseline levels in naïve mice.

Data demonstrating MC1R mRNA expression in spleen and peritonealmacrophages indicated that 30 minutes post-LPS challenge might be theoptimal timepoint for α-MSH or RI α-MSH analog treatment. C57BL/6 micewere injected with LPS, i.p. and 30 minutes later native α-MSH or RIα-MSH analog 891 was administered i.p. at doses ranging from 5 mg/kg to0.156 mg/kg. RI α-MSH analog 891 is an analog of RI α-MSH in which d-Chaand hsiiss D-amino acids were added to the core peptide sequence. The RIα-MSH analog 891 has shown to have increased binding affinity to mouseMC1R and increased potency to stimulate cAMP (data not shown). Resultsshowed significant reduction in TNF-α and IL-10 levels in the nativeα-MSH treated groups compared with PBS vehicle control (FIGS. 16A and16C). MCP-1 was not affected by native α-MSH treatment (FIG. 16B).Treatment of LPS challenged mice with RI α-MSH analog 891 showed similarresults with a significant decrease in TNF-α and IL-10 with no affect onMCP-1 levels compared with vehicle control (FIGS. 16D and 16F). Lowerdoses of both native α-MSH and RI α-MSH analog 891 showed the greatestsuppression of inflammatory cytokines. Dexamethasone positive controlconsistently showed suppression of TNF-α and MCP-1 levels.

Example 18 Stability of Peptides in Plasma

The serum half-life of native α-MSH has been estimated to beapproximately 10 minutes. Despite this limited half-life, the peptide isstill capable of eliciting potent anti-inflammatory activities. However,more stable α-MSH analogs are necessary to increase the potency ofanti-inflammatory activities and be further developed as a therapeutic.A D-amino acid analog of native α-MSH (referred to as retro-inversoα-MSH or RI α-MSH) was synthesized and is more stable than native α-MSH.The D-amino acid form of peptides is more resistant to proteolysis andis metabolized at a slower rate than the L-amino acid form of peptides.

The stability of RI-α-MSH was determined by incubation in plasma or PBSin vitro at 37° C. for 24 hours. Aliquots were taken and proteinsprecipitated with 2 volumes of acetonitrile. α-MSH was used as a controland bradykinin as an internal standard. After centrifugation, sampleswere frozen at −80° C. until LC/MS/MS (MRM) analysis using a C18 columnseparation and positive electrospray ionization mode. As shown in FIG.17a , MSH showed a half-life of about 3 hours in plasma, but was stablein PBS. However, RI-α-MSH was stable both in PBS and plasma, showing nodetectable degradation over 24 hours.

The pharmacokinetic (PK) profiles for α-MSH and RI-α-MSH suggest thatRI-α-MSH has a longer serum half-life than native α-MSH (FIG. 17b ).Mice treated with a single dose of 100 μg RI-α-MSH or α-MSH hadmeasurable serum levels of RI-α-MSH after 24 hrs but there were nodetectable levels of α-MSH 120 minutes post-treatment (n=5).

Example 19 Binding Effects of Retro-Inverso Peptides

A core MSH tetrapeptide containing a D-Phe (HfRW) (variant disclosed inSEQ ID NO:1) is sufficient to produce significant binding to MC1R (20-50nM Ki). However, very little binding is observed with the retro-inversoversion of the same tetrapeptide (wrFh, (variant disclosed in SEQ IDNO:2) 883, Ki>30 uM, Table 1), showing that retro-inverso peptide failsto fully contact the receptor in the same fashion as HfRW (variantdisclosed in SEQ ID NO:1). Addition of fatty acyl groups to theN-terminus of HfRW (variant disclosed in SEQ ID NO:1) selectivelyimproves its binding to MC1R, yielding an affinity comparable to MSH.This is observed for the stearyl-HfRW (variant disclosed in SEQ ID NO:1)peptide at MC1R (1.3 nM, peptide 820). Addition of a diaminohexanestearyl group to the retro-inverso wrFh (variant disclosed in SEQ IDNO:2) (peptide 882) improves binding to MC1R (120 nM, >250-fold) and toa comparable extent as stearic acid addition to the N-terminus of HfRW(variant disclosed in SEQ ID NO:1) (80-fold), but does not achieve theaffinity seen for RI-MSH (4.1 nM), again indicating other residues inRI-MSH play a larger role in binding of RI-MSH to MC1R than with MSH.

Example 20 Stereochemistry Effects on Binding of Retro-Inverso Peptides

The stereochemistry of the residues in the core retro-inversotetrapeptide has a significantly different effect on the binding to MC1Rthan in the L-form MSH. As shown in FIG. 19, inversion of the coretetrapeptide residues in RI-MSH to the L-form (peptides 884, 893-895)all reduced the affinity of the peptide for MC1R. Strikingly andunexpectedly, inversion of the D-Phe to L-Phe in the RI sequence causeda 20-fold reduction in binding, whereas the corresponding change in HFRW(SEQ ID NO:33) (L-Phe to D-Phe) has been reported to improve binding asmuch as 400-fold. For the other residues, stereochemical inversion wasfound to have a lesser effect on RI-MSH than on the core HfRW peptide(variant disclosed in SEQ ID NO:1) again indicating the relativeimportance of each residue in the core tetrapeptide is lower in RI-MSHthan in the HfRW tetrapeptide (variant disclosed in SEQ ID NO:1).

Example 21 Effects of End-Capping Retro-Inverso MSH

Another possible route to achieving higher affinity for MC1R is based onthe observation that end-capping the HfRW peptide with phenyl butyricacid selectively and strongly increases the affinity of HfRW for MC1R(Ki=6 pM). However, no increase in the affinity of a truncated RI-MSHbearing a C-terminal phenylpropylamide (peptide 847) was observed, witha Ki of 150 nM observed for both the peptide truncated at the histidineresidue (peptide 847int) and its aminopropyl-phenyl adduct (peptide 847,Table 1), demonstrating that with RI-MSH, binding is altered in a mannerdisallowing simultaneous interaction of the tetrapeptide sequence andthe postulated aromatic interaction site near the histidine-bindingelement in MC1R.

Example 22 Synthesis of a Toxin Conjugate of Retroinverso MSH

A modified version of peptide 891 (Table 1) in which a cysteine isappended to the N-terminus is generated by Fmoc chemistry. A conjugateof monomethyl auristatin E (MMAE) containing a protease-sensitivevaline-citrulline linker with a maleimido-caproyl moiety for coupling tothiols is synthesized according to Doronina et al. (2003) NatureBiotechnol. 21:778-784, incorporated herein by reference. The peptideand MMAE conjugate are incubated in a solution of 25 mM Na phosphate, 2mM EDTA pH7 for 14 h at 25° C. and the product purified by C18reverse-phase HPLC (RP-HPLC).

Example 23 Effect of RI α-MSH Analogs on cAMP in Murine Melanoma CellLines

Both native α-MSH and RI α-MSH increase cAMP levels in murine melanomacells. RI α-MSH peptide analogs were generated to determine whethermodifications to the core sequence of RI α-MSH peptide would elevatecAMP levels compared with the RI α-MSH peptide. A dose responseexperiment was carried out for the generated RI α-MSH analogs in thecAMP assay using murine B16-F1 melanoma cells.

In Vitro Cell Cultures:

B16-F1 murine melanoma cells were cultured into 96 well plates 5×10⁴cells/well overnight in media with L-glutamine and pen/strep and FBS.Media was removed and new media with IBMX was added to the cells for 1hr. Cells were then treated with RI α-MSH, or RI α-MSH peptide analogs(890, 891, 892, 893, 894 or 895). Cells were lysed after 30 min using acAMP assay kit and supernatant were used in the assay. Forskolin at 10μM served as positive controls.

cAMP Levels:

Intracellular cAMP was measured using a cAMP competition Assay Kit(Amersham Biosciences). All cell lysate samples were diluted 1:100 foranalysis.

Peptides:

869 vpkGwr(d-Cha)hemsys (SEQ ID NO: 63) 872 vpkGwrfremsys (SEQ ID NO:66) 880 vpkGwrFhsiiss (SEQ ID NO: 4) 878 vpkGwrfhe(d-buthionine)sys (SEQID NO: 72) 886 RI-M505 vpkgwrfhsiiss (SEQ ID NO: 76) 890vpkGwr(d-Cha)he(d-Buthionine)sys (SEQ ID NO: 77) 891 vpkGwr(d-Cha)hsiiss(SEQ ID NO: 5) 892 SYSMEH(Cha)RWGKPV (SEQ ID NO: 6) 893 vpkGWrfhemsys(SEQ ID NO: 78) 894 vpkGwRfhemsys (SEQ ID NO: 79) 895 vpkGwrfHemsys (SEQID NO: 80)

Results:

Murine melanoma B16-F1 cells were treated with RI α-MSH, or RI α-MSHanalogs (869, 872, 880, 878, 886 and 890-895) at a concentration rangeof 10⁻⁴-10⁻¹¹ M. cAMP levels from cells treated with RI α-MSH, or RIα-MSH analogs. The majority of the RI α-MSH analogs showed improved EC₅₀values compared with RI α-MSH with the exception of analog 894. Analogs891, 892 and 886 showed the greatest improvement in EC₅₀ values in thecAMP assay compared with the RI α-MSH peptide. In summary, RI α-MSHanalogs showed an improved dose response and EC₅₀ value compared withthe RI α-MSH peptide. See FIG. 22 for graphical data and Table 2 forEC₅₀ data.

TABLE 2 EC₅₀, μM EC₅₀, μM EC₅₀, μM α-MSH 0.0004 α-MSH 0.0004 α-MSH0.0004 RI α-MSH 9.7 RI α-MSH 9.7 RI α-MSH 9.7 Analog ID: Analog ID:Analog ID: 890 3.6 878 0.13 869 0.014 891 0.081 880 0.023 872 2.0 8920.021 886 0.002 878 0.13 893 0.27 894 212 895 0.3

The invention claimed is:
 1. A compound that selectively bindsmelanocortin 1 receptor (MC1R), said compound comprising a coretetrapeptide having the sequence: His Xaa Arg Trp (SEQ ID NO: 1),wherein Xaa is D-Cha or Cha; or a pharmaceutically acceptable saltthereof.
 2. The compound of claim 1, said compound comprising apolypeptide having the sequence: Ser Tyr Ser Met Glu His Cha Arg Trp GlyLys Pro Val (SEQ ID NO: 6).
 3. A pharmaceutical composition comprisingthe compound of claim 1 and a pharmaceutically acceptable excipient. 4.The compound of claim 1, wherein said compound is conjugated to abiologically active moiety.
 5. The compound of claim 1, wherein saidcompound is PEGylated.
 6. The compound of claim 1, wherein said compoundexhibits at least one of the following properties: ability toselectively activate MC1R; stability in plasma in vitro; or resistanceto protease degradation.
 7. The compound of claim 2, wherein saidcompound is PEGylated.
 8. The compound of claim 2, wherein said compoundexhibits at least one of the following properties: ability toselectively activate MC1R; stability in plasma in vitro; or resistanceto protease degradation.
 9. A method of treating an autoimmune diseaseor condition in a subject in need thereof, comprising administering tosaid subject a pharmaceutical composition comprising a pharmaceuticallyacceptable excipient and a therapeutically effective amount of thecompound of claim 1, wherein said autoimmune disease or condition isselected from the group consisting of multiple sclerosis, diabetes typeI, aplastic anemia, Grave's disease, coeliac disease, Crohn's disease,lupus, arthritis, osteoarthritis, autoimmune uveitis and myastheniagravis.
 10. A method of treating inflammation in a subject in needthereof comprising administering to said subject a pharmaceuticalcomposition comprising a pharmaceutically acceptable excipient and atherapeutically effective amount of the compound of claim
 1. 11. Themethod of claim 10, wherein said inflammation is associated with adisease selected from the group consisting of inflammatory boweldisease, rheumatoid arthritis, allergy, atherosclerosis, psoriasis,gastritis and ischemic heart disease.
 12. A method for reducing orinhibiting transplant rejection in a subject in need thereof comprisingadministering to said subject a pharmaceutical composition comprising apharmaceutically acceptable excipient and a therapeutically effectiveamount of the compound of claim
 1. 13. A method of treating melanoma ina subject in need thereof comprising administering to said subject apharmaceutical comprising a pharmaceutically acceptable excipient and atherapeutically effective amount of the compound of claim
 1. 14. Themethod of claim 13, wherein the compound is conjugated to an anti-tumorpayload.
 15. A method of treating an autoimmune disease or condition ina subject in need thereof, comprising administering to said subject apharmaceutical composition comprising a pharmaceutically acceptableexcipient and a therapeutically effective amount of the compound ofclaim 2, wherein said autoimmune disease or condition is selected fromthe group consisting of multiple sclerosis, diabetes type I, aplasticanemia, Grave's disease, coeliac disease, Crohn's disease, lupus,arthritis, osteoarthritis, autoimmune uveitis and myasthenia gravis. 16.A method of treating inflammation in a subject in need thereofcomprising administering to said subject a pharmaceutical compositioncomprising a pharmaceutically acceptable excipient and a therapeuticallyeffective amount of the compound of claim
 2. 17. The method of claim 16,wherein said inflammation is associated with a disease selected from thegroup consisting of inflammatory bowel disease, rheumatoid arthritis,allergy, atherosclerosis, psoriasis, gastritis and ischemic heartdisease.
 18. A method for reducing or inhibiting transplant rejection ina subject in need thereof comprising administering to said subject apharmaceutical composition comprising a pharmaceutically acceptableexcipient and a therapeutically effective amount of the compound ofclaim
 2. 19. A method of treating melanoma in a subject in need thereofcomprising administering to said subject a pharmaceutical comprising apharmaceutically acceptable excipient and a therapeutically effectiveamount of the compound of claim
 2. 20. The method of claim 19, whereinthe compound is conjugated to an anti-tumor payload.